How to Characterize Peptoid Nanosheet Surfaces

Background

Peptoids are protein-like polymers that can replicate the behavior of some biomolecules. In 2010, researchers at Lawrence Berkely National Laboratory in California, USA created the first peptoid nanosheets [1]. Highly organized 2-dimensional structures which can be easily functionalized are extremely important. They are necessary for creating complex nanostructures for a variety of applications such as bio-molecular lithography for nanoelectronics.

In that original study, the authors used a variety of techniques to characterize their nanosheets. First, they used Nile Red dye to stain the peptides and view the nanosheets using a fluorescent optical microscope. They used an AFM to measure the thickness of the sheets produced. They also used an SEM to image individual sheets, and finally they used a TEM to show the individual peptoid chains assembled into a sheet.

For this paper, the researchers did not have access to an AFM-IR instrument, but PiFM and PiF-IR would have been uniquely suited to analyze those samples because of the incredible IR resolution and detection sensitivity offered. With that in mind, consider where PiFM and PiF-IR could have fit into the process. For example, PiF-IR spectroscopy could have been used to verify the chemical identity of the nanosheets by comparing their IR spectrum to FTIR or theoretical models. Furthermore, while the AFM was used to measure the height of the sheets, PiFM could have been used simultaneously to measure how chemically uniform the surface was. If the study focused more on functionalizing the surface, then PiFM and PiF-IR could be used to study the nanostructures created. These are speculations, but instead of imagining an alternate reality, one should look at real data on similar materials to get a concrete understanding of the nano-chemical analysis that is now possible on nanosheets such as these.

The samples described below are a real-world example for how nano-chemical analysis can be used to characterize peptoid nanosheet type materials.

Sample descriptions

The goal was to produce a monolayer of peptoid film as shown in the cartoon below.

The first sample should be a thick, 30 nm film. This will ensure that there is plenty of material for PiFM & PiF-IR analysis which will be helpful for later comparisons.

The second sample should be a monolayer of peptoid film that is produced after the thick film is annealed and washed to remove ungrafted layers that weren’t bonded to the Si substrate.

Nanosheet characterization

Sample 1

AFM topography on the 30 nm thick peptoid film shows some taller topographic features. When PiF-IR spectra are taken at various locations on this surface, the results are very reproducible, and there are multiple peaks that match the FTIR reference spectrum for the peptoid molecules [Figure 2].

There are also some very strong peaks in the PiF-IR spectra that do not appear to be related to the peptoid molecules. In figure 3, the peaks highlighted in brown match with an organic siloxane material very well. The peaks highlighted in blue, are both known to be associated with C-H bending modes [Figure 3].

While PiF-IR spectroscopy is extremely powerful for identifying surface chemistry, the actual surface distribution is best analyzed using PiFM chemical maps.

1672 cm⁻¹ is a strong peak in the PiF-IR spectra that is also associated with the peptoid molecules. By tuning the laser to this wavenumber, and mapping the surface absorption, one can create a PiFM image that shows the spatial distribution of the peptoid molecules [Figure 4].

In this thick sample, the peptoid molecules appear to be distributed evenly, except for where the taller topographic features are. At those locations, the signal is attenuated, suggesting that there may be another material on top.

Sample 2

With the data on sample one as a baseline, one can now turn their attention to the monolayer peptoid film.

Unfortunately, initial spectra taken randomly on this surface do not show any peaks associated with the peptoid molecules [Figure 5]. Instead, they just have the peaks associated with organic siloxane and the C-H bending mode as seen in sample 1. To double check for peptoid molecules, a PiFM image taken at 1633 cm⁻¹ can be used to search for peptoid fragments since they will be highlighted in the PiFM image.

This strategy proved extremely helpful since there are indeed a few different bright spots highlighted in the PiFM image [Figure 6]. PiF-IR spectra on these bright spots also show the amide peak expected for the peptoid molecules! One of the fragments appears quite large, and is visible in the AFM topography. However, some of the spots visible in the PiFM image are not at all remarkable in the topography.

By using a line trace to measure the height of one of these topographic features, we can see that PiFM and PiF-IR analysis was able to identify this peptoid fragment even though it is only 0.5 nm tall [Figure 7]!

If all these pieces are put together, then the complete data set would look something like Figure 8. The PiFM images show the locations of the peptoid fragments and the substrate. The red and purple spectra are taken on the fragments, while the other spectra are on substrate. The black lines mark peaks that correlate with the peptoid FTIR spectrum, the blue lines mark peaks that are associated with a C-H bending mode, and the brown lines mark peaks that are associated with organic siloxane.

Conclusions

Even though neither sample 1 nor sample 2 gave the expected results, using PiFM and PiF-IR to analyze the surface provided invaluable information. On sample 1, PiF-IR spectroscopy detected many peaks associated with other materials besides the peptoids. Also, PiFM chemical maps showed that there are some contaminant particles on the surface above the peptoid layer.

Sample 2 turned out to not be a monolayer at all. Instead, only small fragments of peptoid molecules were left on the surface which were found by searching using a PiFM image. PiF-IR spectra on the fragments agreed well with the spectra taken on sample 1. However, clearly the process for creating a monolayer of peptoids should be re-examined in this case.

References

1. Nam, K., Shelby, S., Choi, P. et al. Free-floating ultrathin two-dimensional crystals from sequence-specific peptoid polymers. Nature Mater 9, 454–460 (2010). https://doi.org/10.1038/nmat2742