Unambiguous Nanoscale Characterization of Self Assembled Monolayer Coverage

Introduction

Surface functionalization via SAM (self-assembled monolayer) coverage is utilized in a range of fields, including biotechnology, semiconductor processes, microfluidics, lab-on-a-chip devices, medical implants, biosensors, and wearable technologies. However, determining the quality and state of surface functionalization is surprisingly difficult given how critical it is. Most analytical techniques with molecular identification capability (such as XPS, Raman, FTIR, water contact angle measurement, and even ToF-SIMS) average over nanoscale heterogeneity. Atomic force microscopy (AFM) and other microscopy techniques with nanoscale resolution are frequently used to assess surface topography, with the expectation that the presence of aggregates or extrusions signifies incomplete or heterogeneous functionalization. However, a uniformly flat microscopy image can be ambiguous, as it may correspond either to an unmodified substrate or to a perfectly flat, fully functionalized monolayer. Thus, image-based analysis alone is insufficient for confirming the quality of SAM coverage.

Current methods

This limitation is exemplified in Figure 1, where two surfaces, each exhibiting ~1 nm of height variation, appear topographically homogeneous with a few particulate clusters. Although both samples have undergone attempted peptoid functionalization, the formation of a monolayer remains uncertain. Based solely on these AFM images, it is not possible to distinguish between a successful and unsuccessful functionalization.

From the AFM alone, it is impossible to tell that only one of these surfaces has a successful monolayer. Photo-induced Force Microscopy (PiFM) overcomes this limitation by simultaneously providing high-resolution topographical imaging and nanoscale FTIR-like PiF-IR spectra with spatial resolution down to 5 nm. This combination of functionalities allows chemical characterization of the surface. This data can be used to differentiate a functionalized surface and a bare substrate.

The AFM image on the right side from Figure 1 was analyzed using our PiFM system. We collected PiF-IR point spectra on the flat regions (gold, orange, purple, and light blue spectra, as show in figure 2). These spectra exhibit repeatable peaks at ~800 cm⁻¹, 1020 cm⁻¹, 1100 cm⁻¹, and 1260 cm⁻¹, indicative of siloxane contaminants, as well as additional peaks attributable to hydrocarbons at approximately 1725 cm⁻¹, 1460 cm⁻¹, and 1370 cm⁻¹. The uniform intensity of these contaminant peaks demonstrates that the contaminants are homogeneously dispersed across the surface. The PiF-IR point spectra acquired on the clusters (green and red spectra) show the amide peak at ~ 1650 cm⁻¹ consistent with the dominant FTIR peak for the peptoid shown in red above the PiF-IR spectra. Therefore, in this sample, the peptoid molecules were not able to form a monolayer but only cluster.

In contrast, as shown in Figure 3, PiF-IR point spectra acquired from the locations marked by triangles on the AFM image on the left consistently exhibit peaks at 1658 cm⁻¹ and 1447 cm⁻¹, as can be seen from the spectra on the right. These signals are characteristic of peptoid functionalization. The high reproducibility of these PiF-IR spectra across the surface confirms the presence of a uniform peptoid layer.

Therefore, these results highlight that surfaces appearing identical by AFM can, in fact, be chemically distinct, with PiFM enabling the detection of both functionalization and localized contamination.

Patterned SAM

Many times, surface functionalization needs to be constrained into nanoscale surfaces as in directed self-assembly, selective ALD, nano-particle functionalization, and etc. In these cases, the analytical technique must not only have monolayer sensitivity but also nanoscale lateral spatial resolution. To demonstrate this capability, the sample from Figure 3 was e-beam patterned to form a line-space pattern with a linewidth of ~100 nm (170 nm pitch). AFM topography of the pattern (Figure 4, left side) shows that the polypeptoid brush monolayer is ~1 nm in thickness. Two PiFM images acquired at 1658 cm⁻¹(peptoid signal) and 113 cm⁻¹(SiO₂/Si substrate signal) are combined to present the chemical map of the pattern.

PiF-IR spectra (Figure 5) obtained from the substrate regions (red, purple, blue and light green triangles) exhibit a prominent peak at 1113 cm⁻¹, characteristic of the underlying SiO₂/Si substrate, with no peptoid-associated peak at 1658 cm⁻¹. Conversely, PiF-IR spectra obtained from the taller regions (blue, dark green, yellow, and dark red) contain the same 1658 cm⁻¹ peptoid peaks. Note the excellent signal noise observed in PiF-IR spectra even from a 1 nm-thick monolayer.

These findings demonstrate that PiFM enables the detection of monolayer of molecules with true nanoscale spatial resolution.

Conclusion

Overall, this work establishes PiFM as a robust and unambiguous technique for simultaneous characterization of surface topography and chemical composition at the nanoscale. By providing direct, spatially resolved chemical mapping, PiFM addresses the limitations of many conventional techniques and represents a powerful tool for the comprehensive evaluation of surface functionalization in advanced materials and bio interface research.

References

1. Nanopatterned Monolayers of Bioinspired, Sequence-Defined Polypeptoid Brushes for Semiconductor/Bio Interfaces, Beihang Yu, Boyce S. Chang, Whitney S. Loo, Scott Dhuey, Padraic O’Reilly, Paul D. Ashby, Michael D. Connolly, Grigory Tikhomirov, Ronald N. Zuckermann, and Ricardo Ruiz, ACS Nano 2024 18 (10), 7411-7423, DOI: 10.1021/acsnano.3c10204
2. https://molecularvista.com/surface-functionalization-webinar-052023/