Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

The overall goal of this procedure is to obtain quantitative information on the nanoscale interactions present between particles in a liquid-based colloidal system. This is accomplished by first preparing a functional colloidal probe by attaching a single particle onto a tipis atomic force microscope cantilever. The second step is to mount the probe onto the atomic force microscope and prepare a liquid cell with a colloidal particle substrate.

Next, the spring constant and sensitivity of the colloidal probe are calibrated thermally using the microscope software. The final step is to use the colloidal probe and measure interactions with the colloidal part particle substrate in liquid for calibration. A direct measurement of probe sensitivity using a MICA substrate in liquid is also made ultimately colloidal probe.

Microscopy can be used to measure the inter particle forces that may be responsible for coagulation kinetics and physical instability of the studied liquid colloidal system. This method can help determine and measure key parameters responsible for the physical stability and coagulation kinetics of various liquid colloidal systems such as those present in pressurized meter dose inhalers, pharmaceutical, intravenous formulations, biological systems, and several other systems Through the very precise measurements of attraction, cohesion, and de cohesion forces of two solid materials. This method can provide a very deep insight into non-scale chemical and physical interactions of these two solids.

This knowledge can be used to predict the stability of pharmaceutical formulations, but also can be applied to very different biological system To probe the affinity of particles or different solids to cells or bacteria. Begin by preparing the colloidal probe using an atomic force microscopy tip, less cantilever, affix the cantilever to a 45 degree angle holder as seen here. The holder supports the cantilever at 45 degrees above the horizontal.

The next step is to place epoxy on the microscope slide to do this. After preparing the epoxy, use a clean spatula to smear a thin layer onto the slide. Using a temporary adhesive, attach the opposite side of the slide to a custom holder designed to slide onto the microscope lens casing.

Return to the microscope with the slide and holder assembly and slide the holder onto the lens casing when complete. The slide with epoxy should be above the cantilever on the sample platform. With everything in place, observe the cantilever through the microscope and approach the platform and cantilever to the epoxy slide.

Once the cantilever acquires a small amount of epoxy, withdraw the cantilever with the epoxy in place. Use the same technique to attach a two to five micron lipid particle at the apex of the cantilever. Now prepare the atomic force microscopy substrate.

Use a 35 millimeter round cover slip and a thermoplastic mounting adhesive. Heat the cover slip on a hot plate equipped with the temperature probe. When the hot plate is at about 120 degrees Celsius, apply the thermoplastic adhesive, turn off the hot plate and wait for it, and the cover slip to cool to 40 degrees Celsius.

At this point, dust the cover slip with a small amount of colloidal particles. When the cover slip has cooled to room temperature, transfer it to a holder and wash it by pipetting over it. A small amount of liquid medium here, two H three H per fluoro PENTANE or HPFP.

Do this several times to remove unattached particles. The next step is to mount the substrate for use in the atomic force microscope or a FM for this, use the bottom half of a liquid cell mount the cover slip with the colloidal particles in the bottom half of the cell. Make sure the O-ring is seated properly to prevent leaking.

At the atomic force microscope, take precautions against leaks. Protect the microscope stage with a transparent hydrophobic sheet. Then place the liquid cell onto the stage.

After attaching the colloidal probe to the scanning head, assemble that onto the A FM.Start the A FM and instrument software and use the scan head adjustment knobs to bring the cantilever tip into focus and align the laser. To align the laser, monitor the laser intensity and maximize its value. Allow the system to equilibrate in air and then use the deflection adjustment knob to bring the deflection to zero or a slightly negative value.

Next, from the master panel window of the software, go to the thermal panel. To find the sensitivity of the colloidal probe, select calculate inverse optical lever sensitivity, and then capture thermal data. When a prominent peak is apparent, stop capturing data.

Click to zoom in on the main peak, then select initialize fit. Followed by fit thermal data, a sensitivity value will be calculated. Follow a similar procedure to determine the spring constant of the colloidal probe.

Once the sensitivity and spring constant have been found, it is time to add the medium to the liquid cell. Use a syringe containing at least two milliliters of HPFP to slowly add two milliliters while ensuring there are no bubbles present around the cantilever. After adding the medium, realign the laser to account for a change in the refractive index.

Allow the deflection value to equilibrate in the HPFP for five to 10 minutes. Then adjust it back to zero. When ready to scan on the master panel, set the initial scan size to 20 microns.

Scan rate to one hertz, scan angle to 90 degrees and set point to 0.2 volts. Approach the sample and scan. When a particle of interest is found, immediately zoom onto it to prevent extended probe interactions with the substrate scan to acquire an image sufficient to locate the particle on the substrate or know the heights of its major features.

Then switch to the force panel in the software, move the red position bar to the highest position. Set the force distance to five microns. The scan rate to 0.1 hertz and the trigger channel to none.

Make a single force measurement and make sure the probe does not contact the substrate from the graph obtained. Right click on the graph window and select calculate virtual deflection line. This will automatically calculate and update the vertical deflection.

Now change the trigger channel to deflection and set the trigger point to 20 nanometers. Set the force distance to one micron. Adjust the scan velocity to 200 nanometers per second appropriate for the forces associated with this particle.

Then conduct the first of two to three single force measurements on completion of the measurement. Go to the force panel and click on review. To open a master force panel, highlight the most recent force measurement and check that under the axis heading only is checked.

Also, change the x axis input field to the separation distance. Then click on make graph. Go to the parameters tab on the master force panel and adjust the value of the inverse optical lever sensitivity until the contact region of the graph is completely vertical.

Note the value return to the masterforce panel. Open the force tab and the calibration sub tab. Then enter the inverse value of the inverse optical lever sensitivity in the field for deflection inverse optical lever sensitivity.

Repeat the force measurement and adjustment of the sensitivity two or three times to ensure the value does not change significantly with the parameters set, check the liquid medium level and the stability of the deflection. Proceed with measurements of force curves or force maps. After measurements are complete, determine the true sensitivity of the probe.

Remove the liquid cell with the substrate and replace it with the liquid cell. With MICA or other hard surface, immerse the MICA in the same liquid medium. Proceed by conducting a force measurement with the colloidal probe using a relatively large deflection so the force curve has a long region of contact.

The software determines the sensitivity using the slope of the contact region. Use the software calculated sensitivity during the analysis of curves obtained with the probe. This topographical image of a particle substrate was achieved using a colloidal probe in two H three H per fluoro pentane.

The image is less defined than those obtained with a sharpened conical tip. The goal, however, is to find a substrate particle for evaluating inter particle interactions. Scans can further focus on the surface of a single particle force mapping with lipid-based particles in two H three H per fluoro.

Pentane can provide both topographical maps of the sample height and an adhesion map conveying the maximum pole force of each individual force curve. Both types of raw data graphs can be viewed in three dimensional representations. Overlaying them produces a three dimensional illustration of the distribution of adhesion forces as a function of the topography.

Dwell force measurements allow the study of the effect of contact mechanics and length of contact on the adhesive forces. Solid lipid particles were used and measurements indicate that adhesive forces increase as a function of time. Using indentation dwell, the adhesive forces plateau using deflection dwell While attempting this procedure.

It is important to equilibrate the liquid media prior to starting measurement to ensure the most accurate results are obtained.