There's a great need for convenient tools to generate nanometer-scale features and objects, particularly in the study of biological interactions with nanostructured materials and surfaces. This protocol enables rapid and homogeneous replication of protein arrays over centimeter-scale areas with nanoscale resolution. This allows us to study mesenchymal stem cell interactions on these surfaces.

These model surfaces allow us to examine controlled interactions between specific material variables and mesenchymal stem cells, providing fundamental material design criteria that will inform future biomaterial designs. To begin the procedure, thoroughly mix the components of the PDMS prepolymer in a weighing boat. Degas the mixture in a vacuum desiccator for 20 minutes or until all gas bubbles have disappeared.

Then, place the silicon master for the PPL array in a four-centimeter Petri dish. Pour the degassed PDMS prepolymer mix over the master until it is fully covered. Degas the PDMS covering the master for another five minutes.

During the degassing, treat a glass slide with oxygen plasma for one minute. After degassing, carefully place the slide on the PDMS with the treated side facing down. Gently press the slide onto the silicon master to remove trapped air and to ensure that a uniform layer of PDMS is sandwiched between the master and the slide.

Transfer the sandwiched PDMS array to another Petri dish with the silicon master on the bottom. Cure the PDMS in an oven at 70 to 80 degrees Celsius for 24 to 48 hours. Then, remove the cured PPL array from the oven and allow it to cool for 15 minutes.

Use a razor blade to carefully trim excess PDMS from the back and sides of the glass slide. Blow away loose PDMS debris with a stream of dry nitrogen gas. It is important to trim excess PDMS from the back and edges of the array to enable an accurate alignment.

This also helps the user to better observe the conditions of the probes under the AFM during lithography. Wedge a razor blade into the corner of the array at a depth of one millimeter and carefully pry the array from the silicon master in a single, continuous lifting motion. Then carefully cut and scrape away 0.5 millimeters of the PDMS at the edges of the array with a razor blade.

Users should be very careful when prying the arrays from the master to ensure good separation and to avoid damaging the probes. This should be done in a single, smooth action without the arrays falling back onto the silicon master. Treat the PPL array with oxygen plasmid for 30 seconds, then drop 20 microliters of deionized water onto the array surface.

If the water does not spread evenly over the entire array, repeat the plasma treatment. Thoroughly dry the plasma-treated array, then use double-sided carbon tape to attach the array to the atomic force microscopy probe holder. Mount the probe holder on the AFM kinematic holder.

To ink the array, apply a 20-microliter drop of a one millimolar MHA solution in ethanol to the array, avoiding contact between the pipette tip and the array surface. Allow the MHA solution to spread throughout the array. Wait for the ethanol to evaporate.

Mount the probe holder with the PPL array onto the AFM, then place a gold substrate in the center of the AFM sample stage. Fix the substrate in place with adhesive tape. To begin the alignment, run the stage controller setup program to reset all axes and angles to a pre-calibrated zero point.

Use the AFM sample stage X and Y-axis control console to move the substrate to the desired printing location. Switch the stage release lever to release the sample stage and activate the force sensors. Allow the sensors to equilibrate for at least 15 minutes, then use the Z-axis controller to raise the sample stage until the array is visually as close as possible to the substrate.

Ensure that the array is within one millimeter of the substrate. Next, open the automatic alignment program. Fill in the angle step, the coarse step distance, and the fine step distance.

Set the spread sheet file path and attach the spread sheet template. Then open the AFM control software and navigate to the spectroscopy section. Configure the scan head Z-axis oscillation to extend 10 micrometers over 100 milliseconds, hold for 250 milliseconds, retract 10 micrometers over 100 milliseconds, and hold for 250 milliseconds.

Start the scan head oscillation and run the automated alignment. Once the software indicates that the alignment has finished, end the alignment process. Inspect the automatically-generated linear fit of the alignment data to determine whether the alignment was successful and to identify the overall optimum angles, then raise the sample stage in 500-nanometer increments until contact between the array and the substrate is observed with the AFM top-view camera.

Click Stop in the AFM control software to retract the array by 10 micrometers. To begin the PPL process, open the lithography section of the AFM control software. Select the Z-modulation operating mode and navigate to the lithography pattern to be imported.

Fill in the size of the pattern to be generated. Set the origin offset to 25 micrometers on both the X and Y axes, then select Modulation Absolute Z position, set the instrument to simplify to two layers, and set the black and white values to five and negative five micrometers, respectively. Click Okay to apply the settings and close the window.

Open the layer editor from the lithography window, and set the pause time. Next, lower the atmospheric isolation chamber onto the AFM and open its control software. Set a relative humidity of 45%a temperature of 25 degrees Celsius, and an atmosphere exchange flow rate of 500 milliliters.

Implement the settings and wait for the desired humidity level to be attained. Then start the lithographic sequence. Upon completion of the lithography, retract the stage by 500 micrometers.

Remove the atmospheric isolation chamber. Lock the sample stage to deactivate the force sensors. Lastly, remove the substrate from the stage for visualization or further processing.

PPL was used to produce large-scale homogeneous patterns of fine array grids or detailed images. The patterns were visualized for optical microscopy by etching the gold not protected by the deposited thiol. Irregular patterning would indicate damaged or missing probes.

MHA arrays printed on gold with PPL were functionalized with fibronectin and incubated with human mesenchymal stem cells. Individual cells adhered to the fibronectin-functionalized arrays, adapting their morphology to the patterns when necessary. This PPL technique can be used to deposit a wide range of protein patterns for cell biology experiments.

A proficient user can generate patterns like those shown in about three hours. Remember that meticulous array preparation is the key to success. The PPL technique is quite generic and can be used for the deposition of compounds ranging from small molecules to polymers and biomolecules, paving the way for a variety of studies in chemistry, biology, and material science.

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