<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

# Nanoscale Probing of the Supramolecular Assembly in a Two-Component Gel by Near-Field Infrared Spectroscopy

Romain Chevigny,[a] Efstratios D. Sitsanidis,[a] Johanna Schirmer,[a] Eero Hulkko,[a, b] Pasi Myllyperkiö,[a] Maija Nissinen,[*a] and Mika Pettersson*[a]

**Abstract:** The design of soft biomaterials requires a deep understanding of molecular self-assembly. Here a nanoscale infrared (IR) spectroscopy study of a two-component supramolecular gel is introduced to assess the system's heterogeneity and supramolecular assembly. In contrast to far-field IR spectroscopy, near-field IR spectroscopy revealed differences in the secondary structures of the gelator molecules and non-covalent interactions at three distinct nano-locations of the gel network. A β-sheet arrangement is

dominant in single and parallel fibres with a small proportion of an α-helix present, while the molecular assembly derives from strong hydrogen bonding. However, at the crossing point of two fibres, only the β-sheet motif is observed, with an intense π-π stacking contribution. Near-field nanospectroscopy can become a powerful tool for the nanoscale distinction of non-covalent interactions, while it is expected to advance the existing spectroscopic assessments of supramolecular gels.

## Introduction

Fourier transform infrared (FTIR) spectroscopy is widely used to probe the secondary structure of proteins and elucidate their conformations and folding.[1-5] Supramolecular gels, especially those consisting of amino acids or small peptides, have gained momentum for the development of soft artificial biomaterials to mimic the extracellular matrix (ECM) for biomedical applications.[6-11] To date, the secondary and higher-order architectures of the gelator molecules are mainly probed by ultraviolet-visible (UV-vis), circular dichroism (CD) and far-field FTIR spectroscopy techniques.[12-19] However, these techniques solely probe the intermolecular interactions of the gelator molecules and higher-order molecular assemblies within the gel network at the macroscale (i. e. in bulk systems). Indeed, the far-field ATR-FTIR spectra of supramolecular gels contain the average of the vibrational bands of the gel system. At the macroscale, the study of the amide I (1600 to 1700 cm⁻¹) and

amide II (1500 to 1600 cm⁻¹) regions in peptide-based gels gives valuable information on the secondary structure of the gelator molecules and, consequently, their organization in fibrils and fibres, which form the three-dimensional (3D) gel network.[17-19]

To decipher the molecular/higher-order assemblies and type of interactions in a supramolecular gel system, overcoming the sub-diffraction limit of light and probing at the nanoscale is crucial. Scattering scanning near-field optical microscopy (sSNOM) is an emerging surface spectroscopy technique that has received increasing interest over the past decade.[20] This method combines the topological study of atomic force microscopy (AFM) with the absorptive properties of infrared (IR) radiation on organic molecules. The IR incident radiation is focused at the apex of the probing tip into a highly concentrated electric near-field, which interacts with the sample. The interferometric recording of the scattered light provides infrared spectra at a spatial resolution approximately equivalent to that of the tip (20 nm).[21-28] sSNOM has been used for the analysis of polymeric thin film surfaces,[24,29-35] imaging of cells,[36-40] viruses[41,42] and neurons[43] and the structural elucidation of protein fibrils.[44-46]

Although the secondary structure of protein fibrils has been successfully studied by sSNOM, in supramolecular gels the self-assembly of the gelator molecules (e.g. secondary structure) and the type of non-covalent intermolecular interactions (e.g. hydrogen bonding and π-π stacking) responsible for self-assembly have not yet been studied at the nanoscale. To address the nanoscale probing of molecular self-assembly and elucidate higher-order architectures, such as inter-fibrillar interactions, we introduce sSNOM studies of a two-component amino acid-based organogel, which has been recently introduced by our group (Figure 1).[47] We demonstrate that the high sensitivity of nanoFTIR can distinguish the type of secondary structural features among two structurally similar gelator

<figure><img src="image_3.png" /></figure>

Supporting information for this article is available on the WWW under
https://doi.org/10.1002/chem.202300155

<figure><img src="image_4.png" /></figure>

© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

<footnote>[a] R. Chevigny, Dr. E. D. Sitsanidis, J. Schirmer, Dr. E. Hulkko, Dr. P. Myllyperkiö, Prof. M. Nissinen, Prof. M. Pettersson
Department of Chemistry, Nanoscience Center
University of Jyväskylä
P.O. Box Jyväskylä, 35, 40014 JYU (Finland)
E-mail: mikaj.pettersson@jyu.fi
maija.nissinen@jyu.fi</footnote>

<footnote>[b] Dr. E. Hulkko
Department of Biological and Environmental Sciences
Nanoscience Center
University of Jyväskylä
P.O. Box Jyväskylä, 35, 40014 JYU (Finland)</footnote>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** From molecular self-assembly to the macroscopic characteristics of the gel. a) Chemical structures of the protected precursor gelator 1 and activated gelators 2 and 3. Irreversibly cleaved Boc- group and reversibly cleaved tert-butyl groups are highlighted in orange and blue, respectively. b) Schematic depiction of the transient assembly mechanism. i) activation of the precursor gelator 1 into the active molecular building blocks 2 and 3; ii) hydrolysis of 2; iii) esterification of 3; iv) hydrolysis of tBuOAc solvent; v) dissipation of the gel network. c) ATR-FTIR spectrum of the bulk gel system in the range of 1475 to 1750 cm⁻¹ showing the main vibrational bands. Red dashed lines indicate the amide I peaks. The P₁*ref*, P₂*ref*, P₃*ref* and P₄*ref* peaks (green dashed lines) refer to the vibrations of the functional groups of interest (C=C aromatic stretching, amide II N–H bending, C=O ester stretching and C=O carboxylic acid stretching, respectively). The amide II region shows the N–H bending of the amide group. d) Topography image of the gel’s fibres on a gold surface (5 × 5 µm, scale bar 1 µm). Single fibre (plain circle), crossing point of two single fibres (dashed circle) and two parallel fibres (dash-dotted circle).</figcaption></figure>

molecules. We also introduce sSNOM as a valuable tool for investigating the heterogeneity of multicomponent supramolecular gel networks in high spatial resolution and overcoming surface analysis constraints.

# Results and Discussion

## Model Gel

In our bimolecular gel system, a chemically active solvent (*tert*-butyl acetate, *t*BuOAc) controls a set of coupled reactions that promote the dynamic assembly event.[47] The two gelator molecules form in situ by the coupled deprotection/protection reactions of the precursor gelator *N*-tert-butoxycarbonyl-L-

phenylalanyl-L-phenylalanine-*tert*-butyl ester (Boc-Phe-Phe-OtBu) 1 (Figure 1a). Gelation occurs through the irreversible deprotection of the Boc- group in the presence of the *tert*-butyl group, triggered by the addition of sulphuric acid in the medium, as it forms the activated building block Phe-Phe-OtBu 2 (Figure 1a/b). The *tert*-butyl ester group of 2 is reversibly deprotected to form the free carboxylic acid counterpart Phe-Phe 3, which reforms to the *tert*-butyl ester 2 through the hydrolysis of the *t*BuOAc solvent. Through hydrolysis, the *t*BuOAc solvent provides the *t*Bu⁺ cation needed to esterify 3 towards 2 and generates the secondary solvent *tert*-butyl alcohol (*t*BuOH), which acts antagonistically against the primary solvent tBuOAc. Indeed, unlike the free acid 3, its *tert*-butylated counterpart 2 is soluble in *t*BuOH, thus leading to the progressive dissolution of the supramolecular network.
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2.** Spectroscopic and structural information of a single fibre. a) Topography image of the formed fibres on a gold surface (3×3 µm, scale bar 1 µm). The orange spot indicates the location of the recorded reference spectrum for b). b) NanoFTIR spectrum of a single fibre recorded at the position marked by the red circle in a). Average of 10 interferograms, 20 ms integration time. A reference spot was taken on the gold surface. P₁, P₂, P₃ and P₄ (green dashed lines) correspond to the vibrational bands of the functional groups of interest (C=C aromatic stretching, N–H amide bending, C=O ester stretching and C=O carboxylic acid stretching, respectively). c) NanoFTIR expansion spectrum of the amide I area (1600–1700 cm⁻¹) indicated by the red dashed lines in b). Experimental spectrum (black curve) and fitting function for each individual peak. Blue fits refer to β-sheet secondary structures and red fit to an α-helix. d) The height profile of the cross-section taken at the position marked by the blue dashed line in a). The obtained features (periodicity and plateau of height) indicate both β-sheet and α-helical structures.[52] e) Schematic representation of the secondary structures adopted by the single fibres.</figcaption></figure>

The hydrolysis/esterification cycles between compounds **2** and **3** control the formation of the supramolecular network.

Interestingly, for this dynamic system to initiate the gelation process, both building blocks **2** and **3** are required (Figure S1).
<figure><img src="image_9.png" /></figure>

Therefore, the supramolecular network consists of two structurally similar entities differing only at their C-terminus (free carboxylic acid and *tert*-butyl ester), which are intimately linked to one another since neither can support gelation individually.

The far-field ATR-FTIR spectrum of the bulk organogel (Figure 1c and Figure S2) shows the characteristic vibrational bands at 1691 cm⁻¹ and 1660 cm⁻¹, which correspond to *β*-sheet and *α*-helical secondary structures, respectively.[²,⁴,⁵] In general, for diphenylalanine-based (Phe-Phe) supramolecular gels, mainly a *β*-sheet secondary structure (both parallel and anti-parallel) is reported, relating to the strength of *π*-*π* stacking and hydrogen bonding interactions.[¹¹,⁴⁷⁻⁵¹] Therefore, in our study, the obtained peaks P₁ (1496 cm⁻¹, C=C aromatic), P₂ (1550 cm⁻¹, N–H amide II), P₃ (1712 cm⁻¹, *tert*-butyl ester of 2) and P₄ (1732 cm⁻¹, carboxylic acid of 3)[²,³] and the amide I region are of primary importance to reveal the heterogeneity in molecular organization and how non-covalent interactions drive the network formation at the nanoscale.

## Nanoscale Packing Modes and Interactions in a Single Fibre

The 3D gel network consists of single fibres entangled into wider bundles (Figure 1d).[⁴⁷,⁵¹] Here, we compare the supramolecular interactions, as observed, in a single fibre, along two parallel fibres and at the crossing point of two fibres by near-field spectroscopy, which is an important tool to identify non-covalent interactions responsible for the molecular assembly at the nanoscale. Indeed, in contrast to far-field ATR-FTIR, it enables the selective recording of the nanoscale IR profile of a distinguished higher-order structure, such as a single fibre. Therefore, we recorded the nanoFTIR spectrum at a specific position along an individual fibre (Figure 2a), which showed the four vibrational bands of interest, as already observed in the bulk material (P₁, P₂, P₃ and P₄ marked by the green dashed lines in Figure 2b). It is reasonable to assume that due to intermolecular interactions, the shifts observed for P₃ and P₄ are insufficient to inverse their positions. Therefore, we assign the lower wavenumber peak to P₃ and the higher wavenumber peak to P₄. A summary of the peak positions is presented in Table 1 (Figure S5a and Table S1), along with the reference positions in the spectra of the synthesized gelators (powder materials, Figure S3 and S4) and the bulk gel, measured by far-field ATR-FTIR. Based on the nanoFTIR data, the lower wavenumbers of the P₁ (C=C aromatic), P₃ (C=O ester) and P₄ (C=O carboxylic acid) bands in the single fibre, compared to the synthesized gelators (powder materials), indicate that both functional groups are involved in the assembly event. The results are in accordance with previously reported data for assemblies of the Phe-Phe motif.[¹¹,⁴⁸⁻⁵¹]

Although the type of interactions is similar at the nanoscale (single fibre/nanoFTIR) and bulk gel ( macroscale/ATR-FTIR), their strength is different, as the wavenumbers for both carbonyl group signals are significantly higher in the single fibre spectrum. This suggests that along an individual fibre, the hydrogen bonding originating from the C-terminal carbonyl groups of 2 and 3 is weaker compared to the bulk material. This

is due to the averaging of the far-field FTIR, which takes all entities and conformations into account. In contrast, at the nanoscale, only the interactions involved in the formation of a single fibre are collected. The downshift of P₄ is much higher than that of P₃, meaning that the carboxylic acid interacts stronger than the ester group. The reason for this is the steric hindrance caused by the bulky *tert*-butyl group and the stronger hydrogen bonding tendency of free carboxylic acids, confirmed by a previously reported study on carboxylic acid complex formation.[⁵²]

A more detailed study of the amide I region (Figure 2c) revealed a convoluted band which gave rise to four distinct peaks upon deconvolution. The minima of the second derivative within the region of interest were used to determine the number of the individual bands for subsequent fitting (see more in Supporting Information). The peak at 1652 cm⁻¹ is attributed to an *α*-helical structure (red fitting curve), while the three peaks at 1627 cm⁻¹, 1669 cm⁻¹ and 1690 cm⁻¹ correspond to parallel and antiparallel *β*-sheet structures (blue fitting curves).[⁴,⁵] In contrast to previous studies on Phe-Phe-based supramolecular materials where mainly a *β*-sheet structure is reported,[¹¹,⁴⁹,⁵⁰] here the calculated distribution, as obtained by Gaussian fitting (bandwidth, assuming the same intrinsic intensity), is 10% and 90% for *α*-helical and *β*-sheet structures, respectively. This highlights the high spatial resolution of nanoFTIR in assessing the structural differences at the nanoscale, compared to the bulk material far-field ATR-FTIR studies at the macroscale.

In addition to spectroscopy recordings, the height profile measurements have also been used to describe the secondary structure of fibres.[⁵³] We obtained the height profile of a cross-section taken at the same spot as the nanoFTIR spectrum (Figure 2d). The presence of a plateau of height indicates an *α*-helix, whereas periodic features reveal *β*-sheet structures. There is some difference in observed height between this measurement and the literature[⁵³] but it can be explained by the difference in the structure of the reported gelators. Thus, the height pattern provides information on the secondary structure. Both secondary structures are present in the 3D gel network, while the orientation of the side chains dictates whether the molecular arrangement of the gelators is parallel or antiparallel in the *β*-sheet motif (Figure 2e).

## Nanoscale Packing Modes and Interactions of Two Parallel Fibres in Contact

The molecular arrangement giving rise to a single 1D fibre originates from *π*-*π* stacking and hydrogen bonding. For higher-order organizations, however, the strength of the interactions and the type of functional groups involved are affected by the spatial orientation and/or entanglement of the 1D fibres within the supramolecular network. For instance, probing the contact point of two parallel fibres along their longitudinal axes (Figure 3a) reveals differences in the peak intensity and position compared to the IR profile of a single fibre. The 3D topography depiction (Figure 3b) of the two-fibre
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3.** Supramolecular arrangement along two parallel fibres. a) Topography image of two parallel fibres on a gold surface (2×2 µm, scale bar 1 µm). The orange spot indicates the location of the recorded reference spectrum for d). b) 3D magnification of the red rectangle in a), showing two parallel fibres. Vertical dashed lines show the distance between the height maxima. The red (L₁) and black (L₂) dashed lines refer to the cross-section height profile measurement in Figure S6. c) Schematic depiction of the near-field measurement. The probed volume includes the contact point of the two parallel fibres. Numerical values are in nanometres. d) NanoFTIR spectrum of the parallel fibres recorded at the position marked by the red rectangle in a). Average of 10 interferograms, 40 ms integration time. A reference spot was taken on the gold surface. P₁, P₂, P₃ and P₄ (green dashed lines) correspond to the vibrational bands of the functional groups of interest (C=C aromatic stretching, N–H amide bending, C=O ester stretching and C=O carboxylic acid stretching, respectively). e) NanoFTIR spectral expansion of the amide I area (1600–1700 cm⁻¹) indicated by the red dashed lines in d). Experimental spectrum (black curve) and fitting function for each individual peak. Blue fits refer to β-sheet and the red fit to an α-helical structure. f) Schematic depiction of the hydrogen bonding interactions in the molecular arrangement of two adjacent fibres in contact.[55]</figcaption></figure>

system depicts the location of the nanoFTIR measurement. The height of the individual fibres is between 20–30 nm as well as the distance between the height maxima of the cross-section. These values correspond to tubular structures with a diameter of 20–30 nm in contact with each other. The lateral dimension is much larger in the image (~60 nm for each individual fibre) presumably due to the tip convolution effect arising from the AFM probing tip. The recorded image is, therefore, a convolution of the actual object and the finite size tip.[54] As the near-field, generated by the oscillation of the tip on the surface, probes at a spatial resolution of ~20 nm, the recording of the nanoFTIR spectrum covers both fibres and their contact point, allowing us to study the spectral differences compared to a single fibre (Figure 3d). The four bands of interest (P₁, P₂, P₃ and

P₄, marked by the green dashed lines) are summarized in Table 1 (see also Figure S5b). The peak corresponding to the aromatic moieties (P₁) is not shifted compared to the ATR-FTIR profile of the gelator molecules **2** and **3** (powder materials). This suggests that the phenyl rings are not involved in stacking interactions along the parallel fibres. However, a small shift of the amide II peak (P₂) is observed, whereas the carbonyl groups P₃ and P₄ show a higher downshift. The results suggest that hydrogen bonding is stronger when two fibres are connected at their longitudinal axes than in a single fibre. As for single fibre, the terminal carbonyl groups (carboxylic acid and ester) show a different shift depending on their steric hindrance and bulkiness.
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 4.** Secondary structure difference at the crossing point of two fibres. a) Topography image of two crossing fibres on a gold surface (3 × 3 µm, scale bar 1 µm). The orange spot indicates the location of the recorded reference spectrum for d). b) 3D magnification of the red circle in a), showing the crossing point of two fibres (scale bar 800 nm). Dashed lines L₁ (red) and L₂ (black) refer to the cross-section height profile measurement in Figure S7. c) Schematic depiction of the near-field measurement. Numerical values are in nanometres. d) NanoFTIR spectrum of the crossing point of two fibres (obtained from the position marked by the red circle in a). Average of 10 interferograms, 20 ms integration time. A reference spot was taken on a gold surface. P₁, P₂, P₃ and P₄ (green dashed lines) correspond to the vibrational bands of the functional groups of interest (C=C aromatic stretching, N–H amide bending, C=O ester stretching and C=O carboxylic acid stretching, respectively). e) NanoFTIR spectral expansion of the amide I area (1600–1700 cm⁻¹) indicated by the red dashed lines in d). Experimental spectrum (black curve) and fitting function for each individual peak. Blue fits refer to β-sheet and the red fit to an α-helix secondary structure. f) Schematic depiction of the hydrogen bonding and π–π stacking interactions in the molecular arrangement of the two adjacent fibres in perpendicular contact.[⁵⁵]</figcaption></figure>

The deconvolution of the amide I region reveals four bands (Figure 3e) with equivalent positions to the single fibre IR profile (1653 cm⁻¹ corresponding to an α-helical structure (red fitting curve) and the peaks 1626 cm⁻¹, 1679 cm⁻¹ and 1691 cm⁻¹ corresponding to parallel and antiparallel β-sheet arrangements (blue fitting curves)). The calculated composition by Gaussian fitting (20% and 80% for α-helix and β-sheet,

respectively) is also qualitatively similar to the single fibre nanoFTIR measurements. Therefore, although the strength of the supramolecular interactions differs with the spatial location of the fibres, the secondary structures are not affected.

A cross-section of each fibre (L₁ (red) and L₂ (black) lines, Figure 3b) was also used to assess the secondary structure. The corresponding height profile (Figure S6) shows the character-
<figure><img src="image_14.png" /></figure>

<table><caption>**Table 1.** P₁, P₂, P₃ and P₄ peak positions (cm⁻¹) in the nanoFTIR spectrum of crossed, parallel, and single fibres, their reference position in the ATR-FTIR spectra of the bulk gel and the synthesized gelators 2 and 3.</caption><thead><tr><th></th><th>Crossing fibres</th><th>Single fibre</th><th>Parallel fibre</th><th>Gelator 2</th><th>Gelator 3</th><th>Bulk gel</th></tr></thead><tbody><tr><td>P₁ C=C stretch. aromatic</td><td>1489</td><td>1488</td><td>1496</td><td>1496</td><td>1497</td><td>1493</td></tr><tr><td>P₂ N-H bend.</td><td>1556</td><td>1557</td><td>1546</td><td>/</td><td>1550</td><td>1550</td></tr><tr><td>P₃ C=O stretch. ester</td><td>1729</td><td>1727</td><td>1720</td><td>1732</td><td>/</td><td>1712</td></tr><tr><td>P₄ C=O stretch. carbox. acid</td><td>1761</td><td>1754</td><td>1743</td><td>/</td><td>1780</td><td>1731</td></tr></tbody></table>

istic plateau of an α-helix, although wider, while no characteristic features of a β-sheet arrangement were observed for the L₁ fibre. On the contrary, L₂ fibre shows a relatively constant height profile. Thus, the proximity of the two fibres seems to hinder the topographical determination of the secondary structure. However, the information could be obtained from the spectroscopic profile. A depiction of the potential interactions responsible for the gelator's assembly mode in this spatial location is given in Figure 3f. A previously reported crystal structure of the dipeptide Phe-Phe[55] clearly shows hydrogen bonding between the carboxylic acid and the amine groups. In addition, interactions involving the amide group are observed.

Structural Differences at the Crossing Point of Two Single Fibres

In addition to the two parallel fibres, we aimed to decipher the supramolecular interactions at the crossing point of two fibres (Figure 4a). The height of each fibre is approximately 20 nm, while the 3D magnification shows that the fibres fuse at the crossing point. Therefore, the near-field resolution (~20 nm) can probe the fibre interactions (Figures 4b and 4c). The nanoFTIR spectrum at this particular point reveals an opposite trend to the parallel fibres (Figure 4d). Indeed, the peak of the aromatic moieties (P₁) of the building blocks are shifted (Table 1 and Figure S5c), showing their involvement in molecular packing. Interestingly, the peak of the bulky tert-butyl group (P₃) is not shifted, in contrast to the free carboxylic acid (P₄). However, the shift is lower compared to the single and parallel fibres. The results indicate that when two fibres interact perpendicularly (crossing point), the phenyl rings of the molecules interact to maintain their secondary structure and are supported by the hydrogen bonding originating from the carboxylic acid groups (Figure 4f).

The deconvolution of the amide I region of the spectrum reveals a deviating trend from the previous spatial locations (Figure 4e). The presence of the four bands attributed to α-helix (red fitting curve, 1651 cm⁻¹), parallel β-sheet (blue fitting curves, 1627 cm⁻¹ and 1670 cm⁻¹) and antiparallel β-sheet (blue fitting curves 1790 cm⁻¹) is also observed at the crossing point, and their band positions are quantitatively similar. However, the calculated relative composition of the secondary structure differs. Indeed, Gaussian fitting yielded 96% of β-sheet structures and 4% of α-helical structures. In contrast to the single and two parallel fibres, the secondary structure at the crossing point of two fibres is altered and is now dominated by a β-sheet arrangement (Table 2).

<table><caption>**Table 2.** Peak positions (cm⁻¹) and composition (%) of the secondary structure at three different spatial locations of the gel network.</caption><thead><tr><th></th><th>Single fibre</th><th>Parallel fibres</th><th>Crossing fibres</th></tr></thead><tbody><tr><td>β-sheet</td><td>1626</td><td>1626</td><td>1627</td></tr><tr><td>β-sheet</td><td>1669</td><td>90%</td><td>1670</td></tr><tr><td>anti-β-sheet</td><td>1690</td><td>1691</td><td>1690</td></tr><tr><td>α-helix</td><td>1652</td><td>10%</td><td>1653</td></tr><tr><td></td><td></td><td></td><td>20%</td></tr><tr><td></td><td></td><td></td><td>1651</td></tr><tr><td></td><td></td><td></td><td>4%</td></tr></tbody></table>

The cross-sections of the two perpendicular fibres and the corresponding height profile (L₁ (red) and L₂ (black) Figure 4b and Figure S7) show a plateau of height for the L₁ fibre (characteristic of the α-helix structure), although shorter than for the single fibre. The height profiles do not give detailed structural information at the crossing point. Similarly to the parallel fibres, this is observed when the two fibres are adjacent. However, the structural information is evident on the spectroscopic profile. The height profile of the L₁ fibre clearly indicates the contact point, at which it interacts with the L₂ fibre. Figure 4f depicts the interactions maintaining the structure at the crossing point. The interpretation is supported by the previously reported Phe-Phe crystal structure,[55] which shows both π-π and CH-π stacking of the phenyl rings.

It is of note that drying conditions alter the fibrous network depending on the process,[56] and hence the vibrational frequencies of the freeze-dried (sSNOM) and room temperature dried (bulk gel) materials may differ. Freeze drying allows to study the closest state to the 3D material network whereas drying at room temperature distorts the fibre arrangement (restructuration over the slow collapse of the material induced by solvent evaporation). The frequencies for P₃ and P₄ in the bulk gel state are lower than those at the nanoscale, presumably due to the difference in drying conditions (see Table 1). Additionally, the sSNOM spectra were taken only at a few specific locations and a much larger data set would be needed for statistically meaningful comparison of frequencies.

Conclusion

Our study aimed to decipher the non-covalent interactions and the corresponding secondary structure of the gel fibres at a nanoscale spatial resolution with infrared spectroscopy. Depending on the spatial location of the fibrous gel network, the spectral differences provide additional insight into the driving forces leading to its formation.

Our observations demonstrate that the formation of a single fibre and the parallel orientation of two single fibres is dictated
<figure><img src="image_15.png" /></figure>

mainly by hydrogen bonding, mainly originating from the carboxylic acid group, since its shift is higher than that of the bulky ester group. In both configurations, the secondary structure is constant, with a significant contribution of β-sheet and a small proportion of α-helix. However, there is a tremendous change at the crossing point of two fibres, where a preference towards π-π stacking with a weaker contribution of hydrogen bonding is observed. The secondary structure is dominated by β-sheet with a negligible proportion of α-helix. Our findings are consistent with the previously reported results on similar supramolecular gels studied as bulk materials by far-field IR and other spectroscopic methods.[11,48,49,51] However, nanoFTIR spectroscopy gives a deeper insight into the heterogeneity of supramolecular systems and increases our understanding of the molecular assembly modes in relation to the features of the supramolecular network at the nanoscale.

The sSNOM, therefore, proved to be a sensitive and reliable technique to study soft supramolecular materials, as it enables the direct spectroscopic assessment of the molecular building block arrangement while overcoming surface analysis constraints.[57] Thus, this technique has the potential to become a routine spectroscopic assessment tool and expand the range of techniques to study soft supramolecular gels.

## Authors contributions

**R.C.:** Conceptualization, Investigation, Formal Analysis, Validation, Writing - original draft, review & editing. **E.D.S.:** Conceptualization, Investigation, Writing - review & editing. **J.S.:** Investigation, Writing - review & editing. **E.H.:** Investigation, Resources, Writing - review & editing. **P.M.:** Investigation, Resources. **M.N.:** Supervision. **M.P.:** Supervision, Funding acquisition.

## Acknowledgements

The authors would like to acknowledge the Jane and Aatos Erkko foundation for supporting the current work.

## Conflict of Interest

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords:** IR spectroscopy · near-field · secondary structure · self-assembly · transient gel

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Manuscript received: January 17, 2023

Accepted manuscript online: March 15, 2023

Version of record online: ■■, ■■
<figure><img src="image_17.png" /></figure>

**Understanding the nanoscale interactions:** The determination of the secondary structure of supramolecular gels usually refers to the macroscopic material. It is shown that the

secondary structure and non-covalent interactions driving the self-assembly vary at the nanoscale depending on the configuration of the fibrous network.

R. Chevigny, Dr. E. D. Sitsanidis, J. Schirmer, Dr. E. Hulkko, Dr. P. Myllyperkiö, Prof. M. Nissinen*, Prof. M. Pettersson*

1 – 10

**Nanoscale Probing of the Supramolecular Assembly in a Two-Component Gel by Near-Field Infrared Spectroscopy**

<figure><img src="image_18.png" /></figure>