Probing the molecular structure at graphite–water interfaces by correlating 3D-AFM and SHINERS

Domain: neuroscience
Published: 2026-01-31
Editorial outcome: minor_revision

Abstract
Water at solid surfaces is key for many processes ranging from biological signal transduction to membrane separation and renewable energy conversion. However, under realistic conditions, which often include environmental and surface charge variations, the interfacial water structure remains elusive. Here we overcome this limit by combining three-dimensional atomic force microscopy (3D-AFM) and interface-sensitive shell-isolated nanoparticle enhanced Raman spectroscopy (SHINERS) to characterize the graphite–water interfacial structure in situ. Through correlative analysis of the spatial liquid density maps and vibrational peaks within ≈2 nm of the graphite surface, we find the existence of two interfacial configurations at open circuit potential, a transient state where pristine water exhibits strong hydrogen bond (H-bond) breaking effects, and a steady state with hydrocarbons dominating the interface and weak H-bond breaking in the surrounding water. At sufficiently negative potentials, both states transition into a stable structure featuring pristine water with a broader distribution of H-bond configurations. Our three-state model resolves many long-standing controversies on interfacial water structure.

Introduction
Water is essential in biology and in technology. Many water-mediated processes depend critically on the structure of solid–water interfaces, such as the electrode–water interface in electrochemical systems, membrane–water interface of biological cells, and the semiconductor–water interface in biosensing and photocatalysis1–4. Bulk water has a well-known configuration in the form of a dynamic H-bond network5. In contrast, the structure of water near a solid surface (interfacial water) has been a subject of intense debates and controversies. Even the description of ideal interfaces, such as pure water next to a crystalline solid surface, is a challenge for state-of-the-art atomistic simulations1,3. In real life, various salt and organic species, either intentionally added or adventitious, inevitably exist in water. If the concentration of these impurities is low, the bulk water structure is expected to remain unaffected5. However, at the interfacial region (within ≈2 nm from a solid surface), even trace amounts of salt or organic species from the bulk may adsorb or accumulate to a high concentration1,6–8. As a result, the solid–water interfacial structure is influenced by several factors, including the steric hard wall effect, hydrophobicity, electric field, and surface adhesion. The large variety of possible molecular species and interactions make the solid–water interface inherently complex.

In the past three decades, many techniques have been developed or implemented to characterize the interfacial water structure6,9–22. These methods can be broadly classified into three categories: interface-sensitive spectroscopies (e.g., sum frequency generation, X-ray absorption, and surface-enhanced Raman)17–19,23,24, scattering methods (using X-ray or neutron)20–22, and real-space imaging by 3D-AFM6,9–16. The results have been interpreted via two distinct models. One assumes that pristine water molecules occupy the interface, as supported by a lack of non-water peaks in spectroscopies or a ≈3 Å liquid layer spacing in 3D-AFM or X-ray/neutron scattering (intermolecular distance of bulk water is ≈3 Å)9,11,13,15,19,20. The other model hypothesizes that adventitious airborne or liquid-born species (likely hydrocarbons) dominate the 1–2 nm interfacial region. This non-pristine model is based on the observation of hydrocarbon-related vibrational peaks in spectroscopies, a 4–5 Å interlayer distance identified by 3D-AFM, or a reduction of water density at the interface by as high as ≈90%, as measured by X-ray or neutron reflectivity6,21,25–27. These discrepancies cannot be attributed to differences in the solid structure, as they occur for many commonly used substrates including Au, graphite, graphene, and self-assembled monolayers (Supplementary Tables 1–4).

In the presence of surface charges or an electric field, the response of interfacial water structure is also highly controversial, with existing reports claiming markedly different degrees of H-bond reconfiguration or change in the interlayer spacing12,16–19,28 (Supplementary Tables 1–4).

The above controversies in interfacial water structure may stem from several factors. One possibility is the variation in sample conditions across different labs, influenced by differences in water purity, preparation methods, and/or ambient conditions. Another factor is the limited amount and type of information that can be extracted from each individual characterization method; these limits make it difficult to accurately determine interfacial structures using any single technique. In this work, we combined the atomic-resolution imaging method 3D-AFM (Fig. 1a, b) with SHINERS16,19,29 (Fig. 1c, d). Among the existing methods to study solid–water interfaces, 3D-AFM and SHINERS are specifically sensitive to the same 1–2 nm thick interfacial liquid region above flat substrates19,30–32. The capability of SHINERS to detect graphite–water interfaces is further verified by the observation of the graphite G band and the strong potential-dependence of the overall signal, as shown later in this article. On the other hand, 3D-AFM captures the local liquid density30,33 without inducing observable substrate deformations (Supplementary Note 1). Therefore, the combination of these two methods enables a direct and reliable integration of the complementary spatial density profiles (from 3D-AFM) and chemical bonding configurations (from SHINERS), thus providing comprehensive information on the interfacial liquid structure.

Among the existing interfacial systems, carbon–water interfaces are crucial for a wider range of applications, including energy storage, electrocatalysis, biosensing, water desalination, and more34–37. Here, we use highly oriented pyrolytic graphite (HOPG) as the model carbon electrode system, which has a flat, homogeneous basal plane surface enabling a reliable comparison of imaging and spectroscopy data. Additionally, the simple mechanical exfoliation method for surface preparation ensures reproducible measurements in different labs. The experiments were carried out independently over a four-year period under different environmental conditions in two locations, Madrid (Spain) and Urbana (IL, USA). A combination of 16 separate sets of 3D-AFM and 4 sets of SHINERS measurements is reported here; each measurement lasted for 1–2 days, during which the potential-dependence and time evolution of interfacial liquids were thoroughly examined across multiple sample spots. A comprehensive analysis of all the data together reveals three types of interfacial configurations: a transient pristine water state with strong H-bond breaking near open circuit potential (OCP) (state 1), a stable hydrocarbon-dominated structure with weak H-bond breaking of water near OCP (state 2), and a stable pristine water state with a broad distribution of H-bond configurations at sufficiently negative potentials (state 3). These states transition between each other due to either time evolution or potential modulation.

Results
Interfacial configurations at OCP
In this work, OCP values for all the liquid electrolytes were similar and determined to be 0.17 ± 0.15 V vs. Ag/AgCl. For both 3D-AFM and SHINERS, results measured within the range of 0–0.4 V vs. Ag/AgCl did not show observable differences. Therefore, potentials within 0–0.4 V vs. Ag/AgCl can be regarded as at or near OCP.

All 3D-AFM measurements were performed on atomically flat terrace sites of HOPG immersed in water or aqueous solution (Supplementary Fig. 1). The HOPG surface remained stable over time and after potential cycles (Supplementary Fig. 2). While step edges exist on the HOPG surface38,39, those sites were not examined by 3D-AFM in this work. For 3D-AFM, we imaged a volume of at least 5 nm × 5 nm × 1.5 nm along x, y (in-plane), and z (out-of-plane) directions at each location, to ensure statistical significance. At OCP, x–z maps of pure water at the HOPG surface reveal multiple discrete layers, where the interlayer spacing evolved from the initial value of ≈3 Å to 4–5 Å within about 1 h (Fig. 2a, b, Supplementary Fig. 3a–c). The 0 min time point corresponds to the time when the first 3D-AFM data frame was acquired at the pristine HOPG–water interface. The observed time-evolution is consistent with previous reports6,25,40. As explained before, ≈3 Å corresponds to pristine hydration layers, while 4–5 Å is likely due to the accumulation of adventitious species whose exact nature remains unknown to date6,25,40. The average force vs. z curves extracted from the x–z maps (Fig. 2a), shown in Fig. 2b, pure water panel, confirmed the same layered structures with damped oscillations as typical for interfacial liquids30. Most of the force curves obtained in this work exhibited at least two peaks under all sample and probe conditions. Whenever more than two peaks were identified, the upper layers showed similar interlayer distances as the 1st to 2nd layer (Supplementary Fig. 4). Therefore, the 1st–2nd layer distance (d12) is a reliable experimental descriptor of the observed interfacial liquid density profile. This distance will be used for statistically significant analysis throughout this work.

For the rest of the article, although full 3D imaging was performed for each AFM result, we only show the individual and average force curves in the figures to avoid redundancy and highlight the evolution of d12.

3D-AFM of electrolyte solutions, including K2SO4 in water (0.01–0.2 M) and KCl in water (0.1 M), revealed similar layered interfacial liquid structures with nearly the same time evolution of d12 (Fig. 2b, Supplementary Fig. 3). These results underline the reproducibility of the observed interfacial structural evolution. Furthermore, the findings are largely independent of the salt species and concentration. We thus rule out the possibility that changes in interlayer spacing are induced by interfacial ion aggregation. This interpretation is consistent with prior reports suggesting that hydration layer spacings are largely insensitive to the identity and concentration of the supporting electrolyte in dilute solutions (<1 M), since the number density of water is much higher than that of the ionic species in these solutions9,11,30,41,42. Note that the distance between the first peak and the substrate exhibits large variations among most of the results, which have been previously observed in aqueous solutions10,14,43 and attributed to the fluctuations in the hydration condition of the end of the AFM probe, rather than the intrinsic liquid structure44. d12, on the other hand, is robustly reproducible and independent of the AFM probe, as reported in refs. 6, 25, 30, 43, and evident from the data shown in Supplementary Figs. 3–14, Supplementary Tables 5, 6.

Additionally, care must be taken to avoid overinterpreting differences in the observed 3D-AFM force curves. Random fluctuations, due to variations in AFM tip condition, environmental factors and instrument noise (considering the two different locations and four-year timespan), can result in various error ranges in the experimental observables. For example, average d12 values within 2.8–3.3 Å were observed for pristine HOPG–aqueous solution interfaces with different ionic species and concentrations (Supplementary Fig. 15). Force oscillation amplitudes, on the other hand, exhibits larger fluctuations between 0.01–0.1 nN (Supplementary Fig. 16). These variations should not be directly attributed to differences in the electrolytes or ageing/cleanliness conditions.

We further interrogated the chemical composition of the HOPG–water interface using SHINERS. As shown in Fig. 1c, d, the substrate for SHINERS consists of Au/SiO2 core/shell nanoparticles deposited on HOPG (Supplementary Figs. 17, 18). Detailed SHINERS experimental procedures and validation of its non-perturbative nature in probing interfacial liquids were reported in our previous publications16,45. The particle–HOPG gap regions (1–2 nm thick) are hotspots for plasmonic enhancement32. When immersed in aqueous solutions, these hotspots are filled by the interfacial liquid species, which contribute to the SHINERS signal19,31. While bulk solution (more than ≈2 nm away from the electrode surface) also gives rise to a small fraction of the Raman signal, its contribution was subtracted to obtain the presented SHINERS spectra (Supplementary Figs. 19, 20).

The confocal Raman spectrum of bulk solution (0.1 M K2SO4 in water) confirmed the standard O–H stretching (νOH) band of water molecules between 3000–3700 cm−1, as well as the absence of adventitious species within the sensitivity limit (Supplementary Fig. 19). In contrast, SHINERS spectra of an electrochemically cleaned HOPG–solution interface at OCP showed graphite G band46,47 at 1580 cm−1, a weak C–H stretching (νCH) peak48,49 centered around 2900 cm−1, and νOH band with a distinct shape compared to the bulk solution (Fig. 2c, 0 min panels). The νCH mode can be attributed to the presence of small amounts of hydrocarbon species at the interface that may be either aliphatic or aromatic in nature50–52. This as-prepared HOPG–solution interface can be viewed as pristine, since the amount of hydrocarbons inferred from the spectra is minute. The absence of hydrocarbons confirms the effectiveness of the electrochemical cleaning methods (details explained in the following sections) in removing the possible hydrocarbons at the original HOPG–solution interface and ligands on the Au/SiO2 nanoparticle surface. After ageing for about 2 h, the νOH band intensity became lower, the νCH peaks became stronger, and a series of peaks emerged in the 1050–1700 cm−1 range, which we assign as different modes of hydrocarbon fingerprints (FP) (Fig. 2c, 132 min panels). Possible origins of the FP modes include C–O stretches, C–F stretches, and aromatic ring vibrations, among others, although precise assignments are still under debate53. The simultaneous decrease of water peaks (νOH peak area) and increase in hydrocarbon peaks (FP and νCH peak areas) (Fig. 2d) confirmed the interfacial accumulation of hydrocarbons that replaced the original hydration layers. This conclusion is consistent with the increase of interlayer distance from ≈3 Å to 4–5 Å over time observed in the 3D-AFM results (Fig. 2a, b).

Remarkably, ageing leads to significant changes in the νOH peak components at the HOPG–solution interface. The νOH band at the pristine interface was decomposed into five peaks, OH-1–OH-5, with increasing peak positions at 3419, 3517, 3600, 3658, and 3698 cm−1, respectively (Fig. 2c, 0 min panel, νOH region, and Fig. 2e). After ≈2 h of ageing, the first three peaks were red-shifted to 3389, 3495, and 3585 cm−1, respectively, while the OH-4 and OH-5 peaks became absent or negligible (Fig. 2c, 132 min panel, νOH region, and Fig. 2e). For both pristine and aged interfaces, OH-2 remained the dominant peak, with area fractions of 63% and 76%, respectively; upon ageing, the OH-3 contribution diminished nearly five-fold (from 19% to 4%), while OH-1 became more prominent, increasing from 14% to 20% (Fig. 2f).

The assignment of νOH peaks, even for bulk water, remains controversial in modern literature, due to the complexity of the H-bond network of water (presence/absence of up to two donors and two acceptors for each water molecule) and the symmetry of the νOH vibration (symmetric vs. antisymmetric)54–56. Here, we do not aim to precisely attribute each νOH peak to a specific H-bond configuration and/or vibrational symmetry, which would be practically unrealistic. Rather, we assign each peak to an approximate corresponding H-bond number (tetrahedral, trihedral, etc.) following existing interfacial water studies17,19,57,58. Within one broad νOH spectrum, the typical consensus is that peak components with higher wavenumbers roughly correspond to lower H-bond coordination numbers17,19,57,58.

Following the above approach, we attribute OH-1 to an H-bond number close to 4 (either four-fold or a combination of four-fold and three-fold coordination), and OH-2 to an H-bond number near 3 (either three-fold or a combination of three-fold and two-fold coordination)19,57–61. The assignments for OH-3 to OH-5 will be discussed later. In comparison, bulk aqueous solution only has two νOH peak components, OH-1 and OH-2 (Supplementary Fig. 19). Compared to the bulk solution, the pristine HOPG–solution interface features a much smaller fraction of OH-1, slightly larger OH-2, and the emergence of OH-3 to OH-5, indicating strong H-bond breaking effects. After ageing, the overall H-bond number of water at the HOPG–solution interface became higher, as evidenced by the increased area fractions of OH-1 and OH-2 and decreased fractions or absence of OH-3 to OH-5. This reveals that the hydrocarbon layers are less effective in breaking H-bonds of nearby water compared to HOPG. The red shift of the peaks after ageing, on the other hand, is likely due to the weaker hydrophobic interaction between water molecules and the dynamically moving, less dense hydrocarbon layers (aged interface) compared to the fixed, densely packed HOPG substrate (pristine interface). In addition, adventitious hydrocarbons contain C–H bonds with small dipole moments, which can enable weak dipolar interactions with the surrounding water62, thus mitigating H-bond disruption effects.

In SHINERS measurements, the active interfacial liquid regions contributing to the signal are sandwiched between the HOPG surface and SiO2 (shell of the Au/SiO2 particles) (Fig. 1d). To rule out the possible role of silica in producing the emergent interfacial νOH peaks (OH-3 to OH-5), we designed and fabricated a control sample, Au film/SiO2/electrolyte/SiO2/Au nanoparticles. As shown in Supplementary Fig. 21, the silica layer is ≈3 nm thick at the active gap region. This sample serves as a well-defined control system that enables Raman enhancement, is graphite-free, and ensures SiO2 is the dominant surface composition in contact with the probed volume of aqueous solution. SHINERS spectra of this control sample at multiple different areas and varying electrode potentials did not exhibit OH-3 to OH-5 peaks (Supplementary Fig. 21), distinct from the results obtained for HOPG/solution interfaces. These data prove that neither the silica shell layer nor the electrical double layer formed on the shell contributed measurably to the SHINERS peaks of the HOPG–water interface.

Combining 3D-AFM and SHINERS results, we propose the overall structure of the HOPG–water interface at OCP, as summarized in Fig. 2g. A clean, as-prepared interface is in state 1, featuring multiple (at least 2–3) water layers separated by ≈3 Å. The interfacial water has strongly broken H-bonds compared to bulk water and a diverse distribution of H-bond configurations. After ageing, the interface evolves into state 2, which is dominated by 2–3 layers of hydrocarbon molecules with 4–5 Å separation. Water is mostly depleted from the hydrocarbon layers. The water molecules above the hydrocarbons have overall higher H-bond coordination numbers than those at pristine HOPG surfaces, yet still lower than that of bulk water.

Electrified graphite–water interfaces: pristine response
After identifying the two distinct states of the HOPG–water interfacial structure, we proceeded to investigate their potential dependence. As is common for graphite and metallic electrodes, positive potentials tend to induce surface oxidation19,63,64. Therefore, to preserve the electrode structure and to enable the generalization to a broad range of electrode–water interface systems, we applied negative potentials to the HOPG electrode. The potential range was typically ≈0 to –2.2 V vs. Ag/AgCl, large enough to examine rich reconfiguration effects of the interfacial structure. At sufficiently negative potentials, the hydrogen evolution reaction (HER) became significant (Supplementary Fig. 22), producing bubbles in the local area that prevented further 3D-AFM and SHINERS measurements. The bubble onset potential varied for different samples and local areas, but it remained in the −1.6 to −2.6 V range (vs. Ag/AgCl).

In previous microscopy or spectroscopy studies of electrified solid–water interfaces, there was a lack of control of the initial interfacial configuration at OCP, as the system could be in either state 1 or state 2, or an intermediate regime12,18–20,28,65. This is likely the key reason why existing literature shows persistent discrepancies regarding the effect of electrode potential/electric field on the interfacial water configuration (Supplementary Tables 1–4). To resolve such controversies, here we first prepared the HOPG–aqueous solution system to state 1, before examining the potential dependence. State 1 was achieved by either limiting the total sample preparation/air exposure time to no more than a few minutes before starting the measurement, or via electrochemical cleaning after the sample was prepared (as discussed later). State 1 was confirmed when d12 was observed to be ≈3 Å in 3D-AFM, or when FP and νCH peaks were either negligible or much weaker than the νOH peak in SHINERS.

We first performed potential-dependent 3D-AFM in a series of aqueous solutions containing either K2SO4 (0.01–0.2 M) or KCl (0.1 M). A total set of 9 independent 3D-AFM measurements were conducted with a starting condition of d12 ≈ 3 Å (state 1) at OCP, as shown in Fig. 3a, b, Supplementary Figs. 5–11. To minimize the possible convolution of ageing to the potential-dependence of the interfacial water structure, the measurements were conducted either quickly (within ≈20 min for each potential scan) to mitigate ageing or in an argon-sealed liquid cell to slow down the ageing process. As shown in Supplementary Figs. 5–10, argon sealing was usually effective in preserving the pristine interface for at least 30 h. All of the obtained force curves, regardless of the measurement location (Urbana or Madrid) and environmental condition, revealed that d12 is remarkably potential independent within at least 0 to −2.2 V vs. Ag/AgCl. While each individual set of 3D-AFM results showed small variations of d12, mostly within 3.0–3.5 Å, the overall average values were always near 3.3 Å at each of the applied potentials (Fig. 3b).

The observed potential independence of interlayer distance might seem counterintuitive, since potential modulations are typically expected to induce changes in the ionic concentration at the interface. However, as discussed earlier, for dilute aqueous solutions (<1 M) at solid interfaces, both our results and a large body of prior publications supported the salt concentration-independence of the ≈3 Å hydration layer spacing9,11,30,41,42. Therefore, as long as water is still the dominant species within the first 1–2 nm from the HOPG surface in the potential range we studied, it is not surprising to observe a constant ≈3 Å interlayer distance.

Although the interlayer spacing is potential-independent, the polar water molecules at the interface are expected to change their orientations and thus the H-bond configurations at different electric fields. We investigated such effects through multiple sets of SHINERS measurements (Fig. 3c, Supplementary Figs. 23, 24). Similar to 3D-AFM measurements, we first prepared the samples to reach state 1 at OCP, as evident from the weak or negligible FP and νCH peaks. As shown in Fig. 3c, hydrocarbon-related modes were absent not only at OCP but also throughout all the applied potentials, confirming that the interface remained pristine during the whole measurement process. The νOH mode consistently exhibited five peak components across all applied potentials. At more negative potentials, the overall νOH band became broader while the height decreased. The total area of νOH remained nearly constant (Fig. 3d), again confirming that the total amount of interfacial water molecules was potential-independent within the sensitivity limit. This is consistent with the potential-independent force curves and d12 in the 3D-AFM results (Fig. 3a, b). Since water molecules are neutral yet polar, an interfacial electric field can induce the reorientation of the molecules without changing their average distance from the electrode surface, thus preserving the d12 and average interfacial density.

We further analyzed the potential-dependence of the individual νOH peaks (OH-1 to OH-5). OH-1 to OH-4 exhibited red-shifts at more negative potentials (Fig. 3e), likely due to Stark effects. In contrast, OH-5 remained at the same position (≈3700 cm−1) at different potentials. The width of all the peaks exhibited certain levels of increase at more negative potentials (Fig. 3f), leading to an overall broadening of the νOH band. Among the five peaks, OH-2 showed the most pronounced increase in peak width. The broadening effects are likely due to the larger variations in the H-bond configurations that contribute to each peak, as a result of the interfacial electric field. For example, the OH-2 peak may consist of at least two configurations, one with two donors and one acceptor (DDA), the other with one donor and two acceptors (DAA)17,59. At a stronger electric field, the difference in the vibrational energy of these two configurations may become larger; in addition, each configuration may be further split into sub-configurations with different vibrational energies resulting from their orientational differences (e.g., parallel vs. vertical to the electric field). These effects can lead to a broadening of the OH-2 peak. We further observed that the area fraction of OH-2 decreased, OH-3 was nearly constant, and OH-1, OH-4, and OH-5 increased at more negative potentials (Fig. 3g, h). The evolution in area fractions also signifies the diversification of H-bond configurations, where some OH-2 structures split into configurations with either larger (OH-1) or smaller (OH-3 to OH-5) H-bond numbers, due to the orientation-dependent free-energy modulations induced by the interfacial electric field.

The higher-wavenumber peaks, OH-3 to OH-5, are expected to correspond to configurations with low H-bond numbers, such as 2-coordinated water, cation-coordinated water, dangling O–H, and non-donor (ND) water, although the exact peak assignments remain highly controversial17,19,57–61. Nevertheless, one special property worth noting is the lack of Stark shift of OH-5 over the large range of potentials, which was consistently observed in each of the 7 potential scans over 3 independent sets of measurements where OH-5 was present (Fig. 3c, e, Supplementary Figs. 23–26). Stark shift at a given electric field, to the first-order approximation, is proportional to the difference in the dipole moment between the ground and excited state along the direction of the electric field66,67. Therefore, the OH-5 configuration likely has a nearly fixed molecular orientation (no significant fluctuation over time) with a difference dipole parallel to the substrate surface (perpendicular to the interfacial electric field), thus having zero Stark effect. Among all the reported weakly coordinated H-bond configurations, there is only one possible state fulfilling this criterion, the antisymmetric OH stretching of ND water. The ND configuration, proposed before in a previous study of Au–water interface18, may exist at the first hydration layer with oxygen pointing up (away from the substrate) and two hydrogen atoms down (in close proximity to the substrate). While the ground state dipole moment is vertical, the difference dipole of the antisymmetric OH stretching is parallel to the substrate surface, resulting in zero Stark shift. We thus assign OH-5 to the antisymmetric νOH of ND water.

Figure 3i illustrates the overall potential-dependent response of the pristine HOPG–water interface, combining results from 3D-AFM and SHINERS. At negative potentials, the originally transient state 1 evolves into a steady state 3. In this process, the interface remains water-dominated, with a constant ≈3 Å hydration layer spacing and the same overall interfacial water density. The H-bond configurations of the interfacial water become more diverse, featuring a simultaneous increase of both the ice-like 4-coordinated structures and a few low-coordinated states, including the monomer-like or gas-like ND water.

Electrified graphite–water interfaces: non-pristine response
We further investigated the influence of negative potentials on the initially non-pristine HOPG–water interface. State 2, featuring d12 ≈ 4–5 Å or FP (or νCH) peak intensity higher than that of νOH, was reached either unintentionally right after sample preparation (due to adventitious airborne or liquid-borne hydrocarbons), or intentionally through >1 h ageing under ambient conditions after the HOPG surface was immersed in the aqueous solution.

Starting from state 2, we conducted a series of 6 potential-dependent 3D-AFM experiments (Urbana and Madrid), with K2SO4 in water (0.01–0.2 M) and KCl in water (0.1 M) as the electrolyte (Fig. 4a, b, Supplementary Figs. 12, 13). All results consistently showed an abrupt decrease of d12 from 4–5 Å to ≈3 Å at negative potentials, where the transition mostly occurred between −1 to −1.5 V vs. Ag/AgCl. After the transition, further decrease of the electrode potential did not induce more changes in d12, which remained at ≈3 Å in all the datasets (Fig. 4a, b, Supplementary Figs. 12, 13). These results suggest that hydrocarbons were removed from the interface and replaced by water molecules at sufficiently negative potentials, and the water-dominated interfacial configuration was stable under the negative polarization conditions.

Right after the potential was changed back to OCP, the force curves remained mostly unchanged, with the same ≈3 Å interlayer distance (Fig. 4a, b, Supplementary Figs. 6, 7, 13a, 14). However, after about 1 to 2 h under OCP condition, with the aqueous solution exposed to ambient air, we observed a transition of d12 back to ≈4–5 Å (Supplementary Fig. 14). These results are consistent with the ageing effects shown in Fig. 2, confirming that the system reached the transient state 1 right after the potential was switched from negative to OCP, before evolving into the steady state 2 over time under OCP.

We also conducted a series of potential-dependent SHINERS measurements for HOPG/0.1 M K2SO4 in water starting from state 2, with results summarized in Fig. 4c, Supplementary Figs. 25–27. In all these results, FP and νCH bands were much stronger than that of the νOH at initial OCP or 0 V (vs. Ag/AgCl) condition, consistent with state 2. At negative potentials, the hydrocarbon FP and νCH bands became weaker and eventually negligible at ≈−1 to −2 V vs. Ag/AgCl (Fig. 4c, d, Supplementary Figs. 25–27). In contrast, the overall intensity of νOH became stronger. After the potential was gradually changed back to 0 V (ramping from −2 to 0 V in ≈1 h), the hydrocarbon-related peaks only slightly recovered, while the overall intensity/area of νOH was largely preserved (Fig. 4c, d, Supplementary Figs. 25, 26). These results further confirmed the transition from hydrocarbon to water at the HOPG–aqueous solution interface at negative potentials (state 2 to state 3), as well as the reversal to the pristine state 1 immediately after the removal of the electrode potential.

As another indicator of the interfacial liquid structure, we recorded the current transients during SHINERS measurements (Supplementary Fig. 28). The water-dominated interface exhibited larger transient currents than those of the hydrocarbon-dominated one, due to the expected higher double-layer capacitance of the pristine interface.

The overall structural transitions among all three observed states of the HOPG–water interface are summarized in Fig. 4h. Despite the distinct interfacial structures in states 1 and 2, they converge to the same state 3 under sufficiently negative electrode potentials. We propose two possible origins of this effect. One is the change of the interfacial free energy. Since water is polar while hydrocarbons are nonpolar, an interfacial electric field will lower the enthalpy of water molecules with the alignment of the dipoles to the field, while the hydrocarbons’ free energy is not affected. When the electrostatic free energy modulation of the water dipoles is sufficiently large, it may overcome the interaction energy between the hydrocarbons and the graphite surface, resulting in the swapping between water and hydrocarbons at the interface. Simplified calculations show that this scenario is plausible (Supplementary Note 2). The other possibility is electrochemical reactions, including hydrogen evolution and direct hydrocarbon reduction. Cyclic voltammetry measurements revealed hydrogen evolution in the measured potential range (Supplementary Fig. 22), which can produce hydrogen bubbles that may propel the hydrocarbons away from the electrode surface. Additionally, direct electroreduction of hydrocarbons which contain C–O, C–F and aromatic rings is also possible68–70.

To conclude, we have resolved the long-standing controversy on the structure of graphite–water interfaces. By combining microscopy and spectroscopy methods, and precisely controlling the initial sample preparation, ageing time, and electrode potential, we identified the hitherto hidden co-existence of three interfacial states. At an open circuit or an early neutral surface, the interface can be in either the pristine, water-dominant state or the non-pristine, hydrocarbon-dominant state. At sufficiently negative potentials, the interface inevitably transitions into a pristine state with a diverse distribution of hydrogen bond configurations, including monomer-like non-donor water. The hydrogen bond configurations at the interface are subject to modulations of three factors: the graphite surface, the hydrocarbon layer, and the interfacial electric field. We have distinguished the contribution of each factor and constructed a universal interfacial configuration diagram under varying surface charge and ageing conditions.

Methods
Materials
Highly oriented pyrolytic graphite (HOPG) with a mosaic spread of 0.8° ± 0.2° (ZYB grade) was obtained from Bruker Corporation.
Madrid: Potassium sulfate (anhydrous, >99%, Sigma-Aldrich) or potassium chloride (≥99.0%, Sigma-Aldrich) and ASTM D1193 Type I deionized (DI) water (resistivity of 18.2 MΩ cm, ELGA Maxima) were used for preparing electrolyte solutions of various concentrations. Glass vials were rinsed with acetone (≥97.0%, Sigma-Aldrich), isopropanol (99.6%, Acros Organics) and DI water before preparing the electrolyte solutions.
Urbana: Potassium sulfate (anhydrous, >99%, Acros Organics) and ASTM D1193 Type I DI water (resistivity of 18.2 MΩ cm; Synergy UV system, Millipore Sigma) were used for preparing electrolyte solutions of various concentrations. Glass vials were rinsed with acetone, isopropanol, and DI water before being used to prepare electrolyte solutions.

3D-AFM
3D-AFM measurements were independently performed in Madrid (Spain) and Urbana (IL, USA) using amplitude modulation mode with similar methodologies. In both cases, the cantilever was photothermally excited at its first resonance frequency. A low-frequency sinusoidal signal (10–100 Hz) was applied to the z-piezo to modulate the tip–sample distance, synchronized with the x-displacement, to ensure a full z-cycle at each x-position. Amplitude and phase signals were recorded as functions of x, y and z. We found that the signal obtained over x–z frames and 3D volumes tends to better represent the overall interfacial structure compared to a series of force–distance curves obtained at a fixed (x, y) point. The latter approach is sensitive to the local variations of the substrate surface, such as the lattice sites, impurities, adventitious adsorbates, etc., and is thus less representative of the overall interfacial liquid configuration.

In Madrid, measurements were performed using a Cypher S system (Asylum Research Inc., USA) with custom-developed 3D-AFM software. In Urbana, a Cypher ES system (Asylum Research Inc., USA) with the AC Fast Force Mapping (ACFFM) module was used. Silicon probes were used for the 3D-AFM measurements: Arrow-UHF AuD (NanoAndMore, Germany) in Madrid, and FS-1500 AuD (Asylum Research) and PPP-NCH AuD (Nanosensors) in Urbana. Cantilever calibration was performed using the contactless Sader method, as implemented in the commercial AFM software as GetReal.

Cantilevers were cleaned using sequential solvent-rinsing followed by UV-ozone treatment. In Madrid, cantilevers were immersed in a mixture (50:50 in volume) of isopropanol (99.6%, Acros Organics) and DI water, then thoroughly rinsed with DI water. In Urbana, cantilevers were sequentially soaked in acetone (≈30 min), isopropanol (≈12 h) and DI water (≈1 h), then dried using argon (6N, Airgas) or a compressed air duster (Office Depot). After drying, UV-ozone treatment was applied. In Madrid, cantilevers were exposed for ≈1 h using a PSD-UV3 system (Novascan Technologies, USA). In Urbana, ≈5 min exposure was performed using a ProCleaner Plus system (BioForce Nanosciences), with the AFM probe positioned ≈5 cm from the UV source.

Additional details on the cantilever calibration values and 3D-AFM parameters are summarized in Supplementary Tables 5–8.

Electrode potential control was implemented using either a three-electrode or a two-electrode configuration (see Supplementary Fig. 29)16,74–76. In both lab setups, freshly cleaved HOPG was used as the working electrode and platinum served as both a quasi-reference and a counter electrode. In the Madrid lab, a three-electrode setup was used with platinum foils (0.1 mm thick, >99.99%, Sigma-Aldrich) connected to a PalmSens4 potentiostat (PalmSens). The platinum foils were cleaned using piranha solution (2:1 H2SO4:H2O2), followed by sonication in DI water for 5 min. In the Urbana lab, a two-electrode setup was employed, using a platinum ring as the combined counter and quasi-reference electrode. The ring was cleaned by sequential rinsing in acetone, isopropanol and DI water, followed by drying with argon or a compressed air duster. Electrode potential was applied and monitored using a Keithley 2450 source meter. Urbana’s Cypher ES featured environmental control of the cell chamber, which was purged with argon for ≈1–2 min and tightly sealed, enabling stable operation over at least 2 days without any noticeable change in liquid volume.

All measurements were performed at room temperature and ambient pressure.

3D-AFM data processing
Established force reconstruction methods77,78 were implemented using custom-written codes to reconstruct the conservative component of the AFM probe–sample interaction force. To enhance the force signal and improve the visualization of interfacial layers, the Madrid lab applied binomial smoothing (range = 2) followed by background subtraction using a single-exponential fit, while the Urbana lab employed a 50-point moving average and a double-exponential background subtraction.

In the Madrid lab’s workflow, a single representative x–z map was selected from each 3D-AFM dataset and analyzed in detail by examining the individual force–distance curves and their average. The substrate position (z = 0) was defined based on the setpoint amplitude. The Urbana lab averaged each x–z map within a 3D-AFM set into a single force–distance curve. Prior to averaging, the force–distance curves were aligned based on the tip–sample separation corresponding to a phase shift of 22° below the free oscillation phase. The substrate position (z = 0) was then defined as the z-position (below the first water layer) where the force value equaled that at the first water layer position, following protocols in ref. 6. The x–z map-averaged force–distance curves were then collectively analyzed, and their overall histogram and average were used to represent the interfacial structure of the entire 3D volume.

Raman spectroscopy
Raman spectroscopy experiments were performed at Urbana. All Raman measurements were carried out using a confocal Raman imaging system (Horiba, LabRAM HR 3D-capable Raman spectroscopy) with a 633 nm excitation laser at 3.5 mW power. The beam was focused onto the sample surface using either a 50× (with numerical aperture (NA) of 0.5) or 10× (NA 0.25) objective. Scattered light passed through a 300 grooves per mm grating and was directed to a Horiba Synapse back-illuminated deep-depletion charge-coupled device (CCD) camera. Raman spectra from 300 to 4000 cm−1 were collected with an accumulation time of 20–30 s, and repeated 3–6 times for averaging unless otherwise specified. A silicon wafer was used for spectral calibration at the beginning or end of the experiment.

A Raman spectrum of bulk 0.1 M K2SO4 aqueous solution (Supplementary Fig. 19) was obtained with the laser focused on a HOPG surface in the absence of Au/SiO2 core/shell nanoparticles. A 50× objective (NA 0.5) and an acquisition time of 20 s with 6 accumulations were used.

The preparation of Au/SiO2 core/shell particles was described elsewhere45 and is briefly explained here. A 10 mL 0.01 wt.% HAuCl4 (≥99.9%, Sigma Aldrich) aqueous solution was first heated to boiling, then 0.07 mL of 1 wt.% sodium citrate aqueous solution (>99%, Alfa Aesar) was added to the boiling solution and kept heated for 30 min. The resulting solution was cooled to room temperature and characterized by a UV-vis spectrophotometer. A characteristic absorption peak at ≈534 nm indicated the formation of ≈60 nm Au nanoparticles (Supplementary Fig. 17a). For silica coating, 3 mL of the synthesized bare Au particles solution, together with 0.04 mL 1 mM APTMS solution (Sigma Aldrich) and 0.32 mL 0.54 wt.% sodium silicate solution (Sigma Aldrich) was heated to 90 °C and kept for 2 h. The solution was then cooled in an ice bath. To remove ligands and other by-products, 3 mL of the as-synthesized particles were centrifuged at 2020 × g, 10 min. After discarding the supernatant, the particles were redispersed in 1.5–3 mL of DI water. This centrifugation and supernatant removal process was repeated up to five times, and the final product was redispersed in 200 µL of DI water. Transmission electron microscopy (TEM) verified the morphology of the Au/SiO2 particles, while cyclic voltammetry confirmed that the silica shell is pinhole-free (Supplementary Fig. 17b, c).

SHINERS measurements were performed using either a commercial EC cell purchased from DEK Research Instrumentation (Supplementary Fig. 29d) or a similar home-made cell. The as-prepared Au/SiO2 core/shell particle solution was drop-casted onto the freshly cleaved HOPG surface and dried in air for 1–2 h. During drying, the surface usually accumulated organic contaminants, which were subsequently removed by negative electrode polarization (electrochemical cleaning). Further, drying often resulted in non-uniform particle distribution, forming coffee ring-like structures. SHINERS spectra were typically collected ≈20–30 µm from the coffee ring’s edge. A sub-monolayer coverage is expected in these areas (Supplementary Fig. 18). An Ag/AgCl reference electrode (BaSi, 3 M NaCl, MF-2052) and a Pt wire counter electrode (CH Instrument, CHI115) were used in the SHINERS measurements.

All measurements were performed at room temperature and ambient pressure.

The collected Raman spectra were processed as follows: cosmic spikes (sharp peaks) were first removed, followed by baseline correction using a manual spline fit. For the OH-stretching region, the bulk spectrum was subtracted (see Supplementary Figs. 19, 20). The resulting OH-stretching spectra were then analyzed using Voigt profile fitting.

Electrode potential calibration
All the results in this work are reported against Ag/AgCl, unless otherwise specified. The potential difference between the Pt quasi-reference and a standard Ag/AgCl reference electrode (BaSi, 3 M NaCl, MF-2052) was obtained by comparing the corresponding equilibrium redox potential of the redox couple Ferri/Ferrocyanide (5 mM) in 0.1 M K2SO4 solution. 0 V against Pt was found to be equal to 0.22 V vs. Ag/AgCl (Supplementary Fig. 30). It is worth noting that the potential of a Pt quasi-reference electrode may not be exactly fixed; variations in the rough range of ±0.1 V may arise but do not affect the conclusions we reached in this work.

Additionally, electrochemical impedance spectroscopy (EIS) measurements of HOPG in 0.1 M K2SO4 solution revealed a minimum differential capacitance at –0.6 V vs. Pt (–0.38 V vs. Ag/AgCl) (Supplementary Fig. 31). While this value may be close to the potential of zero charge (PZC), the exact PZC value may change at different interfacial states, depending on the presence/absence of adventitious hydrocarbons.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The data that support the findings of this study are available from the corresponding authors upon request. Unprocessed spectroscopic data are provided as Supplementary Data 1. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Re: Nature Communication Review25-49442-T “Correlative angstrom-scale microscopy and spectroscopy of graphite -water\ninterfaces” by Bonagiri et al.\nThis is an excellent paper that combines AFM and SHINERS to investigate the hydration layers adjacent to HOPG surfaces\nin a variety of different, but nominally neutral pH, electrolytes. The core of the paper is aimed at resolving ambiguities\nassociated with the observed spacing between hydration layers that have been observed to vary between ~ 0.3 nm and ~\n0.5 nm in different reports in the literature that include different or unspecified preparation conditions. By a combination of\naging studies under open circuit conditions and polarization studies at negative potential the authors attribute the\nobservation of thicker hydration layer to organic contamination that tends to build up with time under open circuit conditions.\nThe authors show that this layer can be removed at negative potentials which is an interesting and helpful\nfinding. Likewise, an important aspect of the work is that the contributions follow from interactions and parallel experiments performed between\ntwo independent laboratories that represent leading practitioners in fast force methods for probing the hydration layers at\nimmersed interfaces. The effectiveness of sealing the cell is suggested by data in the supplement and perhaps more should\nbe made of this in the main text. The authors provide Tables listing the imaging parameters in the supplement that will\ndefinitely help others interested in reproducing this work.\nThe paper is certainly worthy of publication. That said, the authors may wish to consider the following comments prior to\ngoing forward with publication.\n1. In the case of the SHINERS experiments the preparation routine involved allowing the SHINERS particles to dry in the\nambient for 1 to 2 hours. Did the surface become covered with organic contamination under these conditions? If not, does\nthis say something about the difference between gas phase versus condensed phase exposure? Please clarify the history of\nthe SHINERS experiments in this regard.\n2. What is the prospect of intercalation reactions, e.g. proton or one of the other cations, during the excursion to negative\npotentials? Does the hysteresis between the water spectra e.g. the OH-1 opc water layers, Figure 2c, Figure 3c at 0 V versus\nthat broadened and shifting OH-1 following the step to negative potentials in Figure 4C and returning to 0 V, speak to\nan irreversible component in the cleaning process. Could this be related to intercalation. The authors might wish to consider\nXPS analysis of the HOPG to see if the cation insertion is doing something to the HOPG under the conditions considered.\n....After reading the supplement I see Figure 19 nicely addresses the hysteresis question and perhaps this should be\nmentioned in the main text.\n3. Did you track the current transients associated with the potential step experiments? Is the conversion from the organic\ncontaminated state charge dependent? Are the gas bubbles necessary to remove the organic contaminants whereby the\ngas/electrolyte interface surface area serves to scavenge that at the HOPG interface. Do the current transient for the potential\nsteps reveals the presence of the contamination layers? After looking though the supplement I see the voltammograms...but\nthe variation stated in the text are not evident..so the question remains...as to whether or not chronoamperometry of\nvoltammetry can be used to probe for a clean surface.\n4. In the introduction the statement is made that the hydration layer are independent of the identity of the supporting\nelectrolyte even at concentrations in the range of 0.1 to 1 mol/L. This seems very unintuitive to me. In looking at the variation\nin d12 across the figure set, e.g. 0.29, 0.28, 0.33 nm in Figure 2, perhaps the claim of independence of d12 from ion identity\nis too strong a statement and rather the differences are yet to be revealed or understood due to sampling, analysis, entropic\neffects, etc. My sense is the variation evident in the supplement would support this point of view.\n5. In the same spirit, why is the amplitude of the hydration layer oscillations so much smaller (1/2 o 1/3) in the presence of\nions versus the pure water result in Fig 2b. Likewise after removal of the organic layer the amplitude of the hydration layer is\nreduced to ¼.\n6. The potential dependence of the water modes in Figure 3 is very interesting and the authors might to say how\nreproducible the effects is when you cycle the potential over hours (as the experiments are reported to have taken place over\na span of two days in the cypher ES (....after looking at the supplement this question is nicely addressed please add a\ncomment to main text that points to this element of robustness for the sealed cell...this aspect is presently “hidden” in the\nsupplement). How sensitive is the shift in the water modes to the identity of the supporting electrolyte. The results are shown\nfor K2SO4 but what about the results for KCl or NaCl? Is the same slope obtained in the OH-1 peak position?\n7. Why is the scatter band in Figure 2b for water layers KCl appear to be so much smaller than K2SO4. Is the difference\nsignificant, one might imagine that the halide double layer might organize in a more isotropic fashion…with regard to the\nwater solvation and hydration layers?\n8. This is very useful paper certain to help improve the robustness of the applied methods and thereby suitable for timely\npublication.\n9. The data in the supplement clearly point to the advantages of using the closed ES cell.\n10. The idea of using a nominally outer sphere redox couple to define the value of a quasi-reference electrode is wrong. The\npresence of the redox couple pins the Pt electrode at the E1/2 potential of the redox couple...which is different from the value\nof the Pt floating in a K2SO4 solution. On the scale of the potential changes examined this is a minor point..although when\nH2 is generated in the cell this will tend to drive the Pt reference to H+/H2 potential that is also subject to pH shear in the\nnon-buffered electrolyte. A better way to establish the quasi-ref potential would be too compare the cyclic voltammetry in\nK2SO4 using a Ag/AgCl/Cl vs Pt and trying to minimize the extension into the HER region.", "reviewer_2": "This study explores the complex structure of interfacial water under realistic conditions by employing a combined approach\nof three-dimensional atomic force microscopy (3D-AFM) and interface-sensitive Raman spectroscopy (SHINERS). The\nfocus is on the graphite–water interface, which is highly relevant to applications in biology, energy conversion, and\nenvironmental technologies. The authors identify three distinct interfacial water states: (1) a transient pristine water state\nnear open circuit potential (OCP), characterized by strong hydrogen-bond (HB) breaking; (2) a hydrocarbon-dominated state,\nalso near OCP but more stable, showing weak HB breaking due to the accumulation of hydrocarbon species; and (3) a\nstable pristine water state at sufficiently negative potentials, exhibiting a broader distribution of\nHB configurations. The complementary use of 3D-AFM for spatial liquid density profiling and SHINERS for chemical structure identification offers in\nsitu, high-resolution insights into the interfacial water structure within ~2 nm of the surface. While the study presents a\ncompelling framework, several important points need clarification before the manuscript can be recommended for\npublication:\n1. The authors should present AFM images of the graphite surface and clearly indicate where the measurements were\ntaken. Given that graphite surfaces can include step edges, terraces, and other defects—with known variations in adhesion\nproperties—it is essential to specify whether the measurements were conducted on atomically flat terraces or at defect sites.\nRelevant prior studies, such as H. Lee et al., ACS Nano 9, 3814–3819 (2015) and Z. Ye et al., Appl. Phys. Lett. 106, 231603 (2015), should be cited to contextualize this variability.\n2. To rule out the influence of potential adsorbates formed during electrochemical measurements, the authors should provide\nAFM images of the graphite surface taken both before and after the experiments. This would help confirm that the observed\ninterfacial structures are intrinsic rather than artifacts of surface modification.\n3. The mechanical pressure exerted by the AFM tip during 3D-AFM measurements is expected to be significantly higher\nthan that in SHINERS. The authors should discuss whether this difference in applied pressure could influence the water\nstructure near the interface and whether it could account for any observed discrepancies between the two techniques.\n4. The observed shift in interlayer spacing from ~3 Å to 4–5 Å over the course of one hour warrants further explanation. First,\nhow is the “0-minute” time point defined? Since time is required for probe alignment and approach, there may be an earlier,\nunobserved state preceding the nominal start of measurement. Second, considering the layered nature of graphite, tip-\ninduced deformation or water intercalation between the top and sub-surface graphene layers may contribute to this\nevolution. The authors should clarify the physical origin of the time-dependent changes in interlayer spacing and discuss the\npotential role of tip-sample interactions or interfacial water penetration.", "reviewer_3": "This is an exciting fundamental study of the water/solid interface using a combination of three-dimensional atomic force\nmicroscopy and interface-sensitive Raman spectroscopy. (3D-AFM and SHINERS) It is used to probe of dynamic hydrogen\nbonding network at the interface to understand the role of salt and organic species within the interfacial region within 2 nm of\ngraphite surface. They observe two interfacial configurations, transient state pristine water with strong hydrogen bonds, and\nanother, with hydrocarbons dominating the interface, and weak hydrogen bond breaking. As far as I can tell the procedures\nare reasonable, though I am not an experimentalist. The insight of the observations motivates exploration of\ntheoretical and simulation models of the phenomena observed in this complex system. Communication of the details and findings will\nbenefit the community. Please publish.", "reviewer_4": "This paper studies the interfacial water structure on the HOPG using a combination of 3D-AFM and SEIRAS. While 3D-AFM\nhas greatly advanced our understanding of the liquid-solid interface by providing visualization of the interfacial molecule\narrangement, it offers limited information on the physicochemical interactions between them. This paper provides\na comprehensive picture of the interfacial water structure on both pristine and hydrocarbon-adsorbed HOPG. The\ncorrelative analysis of 3D-AFM and SEIRAS bridges the gap between two contrasting models of the HOPG-water interface:\nmajorly water at the surface and majorly adventitious non-water molecules at the surface. They demonstrate a time-and potential-\ndependent transition between two states. In addition, they report disrupted hydrogen bonding (HB) at the pristine HOPG-\nwater interface, whereas the hydrocarbon-covered surface shows less broken HB. Given that the crucial roles of interfacial\nwater in physics, biology, and especially chemistry as solvent, reactant, and proton donor to modulate the activation energy of\nchemical reactions, this well-written paper will be of broad interest to the readership of Nature\nCommunication. The high-quality correlative approach could serve as a valuable framework for\nfuture studies of interfacial water structures. We therefore support publication of this manuscript after the authors address the following minor comments.\n1. The authors conclude that pristine HOPG, rather than adsorbed hydrophobic molecules (probably adventitious\nhydrocarbons), weakened the HB network of water molecules leading lower HB-bonded or non-HB-bonded water\nmolecules. Since HOPG itself is a carbon-based materials, what kinds of characteristic of HOPG may contribute to this\ndifference?\n2. The authors should discuss potential zero charge (PZC) of the HOPG? The direction and the strength of the interfacial\nelectric field should be discussed with reference to the PZC (rational potential scale).\n3. I recommend against defining ‘0-0.4 V as OCP’. This is an enormously broad potential range and should not collectively\nbe called OCP.\n4. The SHINERS particles will form their own double layer which will potentially be influenced by changes in the charge\ndistribution imparted by the application of different potentials to the working electrode. The authors point to other references\nthat discuss the non-interaction of these particles. However, these discussions need to be present in the current\nmanuscript to make the data credible. Thus, relevant discussion needs to be added.\n5. The authors mention that minimal changes are observed with the presence of ions but Figure 2b shows considerable\nchanges in the force curves. These changes need to be more clearly discussed and elaborated.\n6. How do the authors distinguish hydrocarbon adsorption from potential slow formation of oxygen groups on the HOPG\nsurface that could also lead to structural changes?\nIt would have been nice to show measurements where organics are purposefully added to the electrolyte to corroborate the\nclaim that the changes in force curves occur because of organics adsorption. I realize, however, that this may be beyond the\nscope of the study.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Title: Correlative angstrom-scale microscopy and spectroscopy of graphite–water interfaces\nManuscript ID: NCOMMS-25-49442-T\nAuthors: Lalith Krishna Samanth Bonagiri, Diana M. Arvelo, Fujia Zhao, Jaehyeon Kim, Qian\nAi, Shan Zhou, Kaustubh S. Panse, Ricardo Garcia, and Yingjie Zhang.\nReply to the Editor & All Reviewers\nWe would like to thank the editor for handling our manuscript and for the valuable suggestions on\nformat changes. We have now thoroughly addressed all the reviewers’ comments, revised/added\nsome texts in the main manuscript, and added Supplementary Note 1 and Supplementary Figs. 1,\n2, 15, 16, 28, 30b, and 31 to the Supplementary Information. In addition, we have added “Data\navailability”, “Acknowledgements”, “Author contributions”, and “Competing interests” sections\nat the end of the main manuscript. To facilitate the broad dissemination of our results, we have\nattached an Excel file containing all the source data presented in the main manuscript and\nsupplementary information, and another Excel file containing all the raw Raman spectral data\n(directly produced by the Raman instrument, before any data processing step). We have also\nattached the required reporting summary form. Point-by-point responses to the reviewers’\ncomments are provided below. We believe this manuscript is now at a publication-ready level.\nReply to Reviewer 1\nReviewer comment 1.0: This is an excellent paper that combines AFM and SHINERS to\ninvestigate the hydration layers adjacent to HOPG surfaces in a variety of different, but nominally\nneutral pH, electrolytes. The core of the paper is aimed at resolving ambiguities associated with\nthe observed spacing between hydration layers that have been observed to vary between ~ 0.3 nm\nand ~ 0.5 nm in different reports in the literature that include different or unspecified preparation\nconditions. By a combination of aging studies under open circuit conditions and polarization\nstudies at negative potential the authors attribute the observation of thicker hydration layer to\norganic contamination that tends to build up with time under open circuit conditions. The authors\nshow that this layer can be removed at negative potentials which is an interesting and helpful\nfinding. Likewise, an important aspect of the work is that the contributions follow from interactions\nand parallel experiments performed between two independent laboratories that represent leading\npractitioners in fast force methods for probing the hydration layers at immersed interfaces. The\neffectiveness of sealing the cell is suggested by data in the supplement and perhaps more should\nbe made of this in the main text. The authors provide Tables listing the imaging parameters in the\nsupplement that will definitely help others interested in reproducing this work.\nThe paper is certainly worthy of publication. That said, the authors may wish to consider the\nfollowing comments prior to going forward with publication.\nResponse 1.0: We would like to thank the reviewer for the thoughtful and constructive comments.\nWe highly appreciate the recognition of the significance of our work and careful reading of the\nmanuscript and supplementary information. The reviewer’s insights have been very helpful in\nclarifying and strengthening the presentation of our results. We have carefully considered the\nsuggestions and have made the necessary revisions to the manuscript accordingly. Please find our\ndetailed point-by-point responses below.\nReviewer comment 1.1: In the case of the SHINERS experiments the preparation routine involved\nallowing the SHINERS particles to dry in the ambient for 1 to 2 hours. Did the surface become\ncovered with organic contamination under these conditions? If not, does this say something about\nthe difference between gas phase versus condensed phase exposure? Please clarify the history of\nthe SHINERS experiments in this regard.\nResponse 1.1: The question involves both the SHINERS sample preparation routine and broader\ncontamination mechanisms. Our answers are:\n1) Surface condition after ambient drying in SHINERS experiments. In SHINERS\nmeasurements, including both our work and reports in the existing literature (e.g., ACS Nano\n2013, 7, 8940–8952), the surface commonly becomes covered with organic contaminants after\nthe drying step. This contamination is immediately evident in the Raman spectra, which\ndisplay characteristic C–H stretching bands (2800–3000 cm−1), as well as C–C and C=C\nmodes typical of adventitious carbon (Fig. 4c and Supplementary Figs. 25a,b and 26a,b.). It\nis challenging to control the contamination mechanism in this drying step, as this process does\nnot correspond to either pure gas-phase or pure liquid-phase exposure; during evaporation, a\nthin water layer persists on the surface, creating a semi-wet state. Instead of trying to control\nthe contamination source during drying, our strategy was to “initialize” the sample condition\nafter the SHINERS sample has been prepared. As discussed in Page 8 | Lines 7–10 of the\noriginal manuscript (Page 8 | Lines 23–26 after revision), we applied reductive (negative)\npolarization to clean the surface, as the adsorbed organic species were removed under these\nconditions. After this initiation procedure, we restored a pristine graphite–water interface. All\nthe following air exposure and electrode potential application history were well documented\nin our data and reported in the manuscript (Figs. 2c, 3c, and 4c, Supplementary Figs. 23–27),\nensuring the results correspond to known experimental conditions and can be reproduced by\nothers.\n2) Gas-phase vs condensed-phase exposure: context from prior work. This question relates\nto the broader issue of contamination pathways under gas- versus liquid-phase exposure.\nHaitao Liu’s group has examined these differences extensively, combining electrochemical\nmeasurements with ellipsometry and contact angle analysis (Carbon 2018, 134, 464–469; Nat.\nMater. 2013, 12, 925–931). Their studies demonstrated that both direct ambient air exposure\nand air exposure of the solid immersed in aqueous solution/water (in the time scale of minutes)\nare sufficient to generate measurable contamination layers on the surface. The determination\nof the exact kinetics of the gas-phase vs liquid-phase air exposure is beyond the scope of this\nwork.\nChanges to the manuscript: In the “Methods” section of the main manuscript, we further clarified\nthe electrochemical cleaning procedures for SHINERS sample preparation (Page 18 | Lines 9–10):\n“During drying, the surface usually accumulated organic contaminants, which were subsequently\nremoved by negative electrode polarization (electrochemical cleaning).”\nReviewer comment 1.2: What is the prospect of intercalation reactions, e.g. proton or one of the\nother cations, during the excursion to negative potentials? Does the hysteresis between the water\nspectra e.g. the OH-1 topwater layers, Figure 2c, Figure 3c at 0 V versus that broadened and\nshifting OH-1 following the step to negative potentials in Figure 4C and returning to 0 V, speak to\nan irreversible component in the cleaning process. Could this be related to intercalation. The\nauthors might wish to consider XPS analysis of the HOPG to see if the cation insertion is doing\nsomething to the HOPG under the conditions considered. ....After reading the supplement I see\nFigure 19 nicely addresses the hysteresis question and perhaps this should be mentioned in the\nmain text.\nResponse 1.2: In graphite, the potentials required for intercalation of protons or other cations\n(e.g., Li+ /Na+ / K+ at –2.5 V to –3 V vs. SHE) lie beyond the stability window of water (see results\nin Sci. Rep. 2016, 6, 28421; Adv. Sci. 2017, 4, 1700146). As a result, in aqueous electrolytes cation\nintercalation into HOPG is generally not observed. Consistent with existing studies, in our cyclic\nvoltammetry measurements we do not detect any peaks—either reversible or irreversible—that\nwould be indicative of cation or proton intercalation (see Supplementary Fig. 22).\nAs the reviewer mentioned, Supplementary Fig. 19 in the original manuscript (Supplementary Fig.\n23 after revision) proves that the hysteresis in the 0 V spectra is not due to any other reason except\nthe hydrocarbon removal/accumulation effects. To emphasize this point, we have added an\nadditional comment in the main manuscript referring to this figure.\nChanges to the manuscript: In the “Electrified graphite–water interfaces: non-pristine\nresponse” section of the main manuscript, we added (Page 14 | Lines 23–26): “Additionally, the\nchange in the initial and final 0 V spectra in Fig. 4c cannot be explained by other potential-induced\nhysteresis effects besides the change of hydrocarbons, as the pristine, water-dominated interface\ndid not exhibit any hysteresis effect (Supplementary Fig. 23).”\nReviewer comment 1.3: Did you track the current transients associated with the potential step\nexperiments? Is the conversion from the organic contaminated state charge dependent? Are the\ngas bubbles necessary to remove the organic contaminants whereby the gas/electrolyte interface\nsurface area serves to scavenge that at the HOPG interface. Do the current transient for the\npotential steps reveals the presence of the contamination layers? After looking though the\nsupplement I see the voltammograms...but the variation stated in the text are not evident. So the\nquestion remains...as to whether or not chronoamperometry of voltammetry can be used to probe\nfor a clean surface.\nResponse 1.3: We agree that this is an important point, and we did in fact track the current\ntransients associated with the potential step experiments in both Urbana and Madrid laboratories.\nIn both labs, potential steps performed on hydrocarbon-contaminated HOPG/solution interface\nproduced markedly smaller current responses than those on a pristine interface (see the added\nSupplementary Fig. 28). The pristine interface consistently exhibited larger current transients than\nthose of the contaminated one, revealing that the former has higher double layer capacitance than\nthe latter. This is likely because the hydrocarbons accumulated at the HOPG–solution interface\ninduce a larger spatial separation between the electrical double layers and the HOPG surface,\nwhich further leads to smaller capacitance. Overall, these observations suggest that the transient\ncurrent may indeed serve as a qualitative probe of surface cleanliness. However, more systematic\nmeasurements will be required to establish the current–cleanliness relationship quantitatively,\nwhich is beyond the scope of this work.\nRegarding removal of hydrocarbons, we note that our AFM and SHINERS measurements were\nlimited to potentials below the onset of visible bubble formation, since strong gas evolution would\ndisrupt stable 3D-AFM imaging and SHINERS spectroscopy. This was discussed in the original\nmanuscript (originally Page 7 | Lines 42–45, now Page 8 | Lines 12–15) “At sufficiently negative\npotentials, hydrogen evolution reaction (HER) became significant (Supplementary Fig. 22),\nproducing bubbles in the local area that prevented further 3D-AFM and SHINERS measurements.”\nSince hydrocarbon removal was observed using 3D-AFM and SHINERS measurements, we can\nrule out the possibility that large, visible bubble formation led to hydrocarbon removal. However,\nwe cannot rule out the possibility that small bubbles or hydrogen molecules (formed via hydrogen\nevolution reaction) can be (partially) responsible for hydrocarbon removal. We have discussed a\nfew possible origins for the potential-induced hydrocarbon removal mechanisms in the paragraph\nright before “Conclusion and outlook” in the original manuscript, which we believe is sufficient\nfor the scope of this work.\nChanges to the manuscript:\n1) We added a paragraph in the main manuscript to explain the current transient measurements\n(Page 14 | Lines 28–31): “As another indicator of the interfacial liquid structure, we recorded the\ncurrent transients during SHINERS measurements (Supplementary Fig. 28). The water-dominated\ninterface exhibited larger transient currents than those of the hydrocarbon-dominated one, due to\nthe expected higher double layer capacitance of the pristine interface.”\n2) We added a figure in the Supplementary Information, as reproduced below:\nSupplementary Fig. 28 | Current transients\nrecorded during SHINERS measurements\n(August 10, 2021, Urbana). Gas\nenvironment: air, open. Potential reference:\nAg/AgCl. The pristine and non-pristine data\nwere recorded during SHINERS\nmeasurements shown in Supplementary Fig.\n23 (potential cycle 3) and Supplementary Fig.\n26 (potential cycle 1), respectively. The\nelectrode potential was changed at a step of –\n0.2 V, and was held constant (at the values\nspecified on each panel) while the current\ntransient was recorded. The pristine interface\nconsistently exhibited larger current\ntransients than those of the non-pristine one,\nrevealing that the former has higher double\nlayer capacitance than the latter. This is likely\nbecause the hydrocarbons accumulated at the\nHOPG–solution interface induce a larger\nspatial separation between the electrical\ndouble layers and the HOPG surface, which\nfurther leads to smaller capacitance. Overall,\nthese observations suggest that the transient\ncurrent may serve as a qualitative indicator\nof surface cleanliness.\nReviewer comment 1.4: In the introduction the statement is made that the hydration layer are\nindependent of the identity of the supporting electrolyte even at concentrations in the range of 0.1\nto 1 mol/L. This seems very unintuitive to me. In looking at the variation in d12 across the figure\nset, e.g. 0.29, 0.28, 0.33 nm in Figure 2, perhaps the claim of independence of d12 from ion identity\nis too strong a statement and rather the differences are yet to be revealed or understood due to\nsampling, analysis, entropic effects. My sense is the variation evident in the supplement would\nsupport this point of view.\nResponse 1.4: We fully agree that the error bars of experimental results need to be thoroughly\nanalyzed, before any conclusion on the electrolyte-dependence can be reached. Thanks to the\nsignificant amounts of data we have generated, we have been able to thoroughly quantify the\naverage values and error bars of d with different ion types and concentrations. As shown in the\n12\nadded Supplementary Fig. 15, we can conclude that d is insensitive to the supporting electrolyte\n12\nwithin the error range of our experimental results. Of course, below the experimental error range\n(roughly 0.2 Å), we cannot make any statement about the electrolyte-dependence.\nChanges to the manuscript:\n1) The original statement in the main manuscript Page 4 | Lines 22–23, “This interpretation is\nconsistent with prior reports that showed the salt-independence of hydration layer spacings in\ndilute solutions (<1 M)”, is now changed to (Page 4 | Lines 28–31) “This interpretation is\nconsistent with prior reports suggesting that hydration layer spacings are largely insensitive to the\nidentity and concentration of the supporting electrolyte in dilute solutions (<1 M), since the\nnumber density of water is much higher than that of the ionic species in these solutions”.\n2) We added a new paragraph in the main manuscript to further clarify the error analysis and\npotential origins of experimental errors (Page 4 | Lines 39–46): “Additionally, care must be taken\nto avoid overinterpreting differences in the observed 3D-AFM force curves. Random fluctuations,\ndue to variations in AFM tip condition, environmental factors and instrument noise (considering\nthe two different locations and four-year time span), can result in various error ranges in the\nexperimental observables. For example, average d values within 2.8–3.3 Å were observed for\n12\npristine HOPG–aqueous solution interfaces with different ionic species and concentrations\n(Supplementary Fig. 15). Force oscillation amplitudes, on the other hand, exhibits larger\nfluctuations between 0.01–0.1 nN (Supplementary Fig. 16). These variations should not be directly\nattributed to differences in the electrolytes or ageing/cleanliness conditions.”\n3) We added Supplementary Fig. 15:\nSupplementary Fig. 15 | Summary of\ninterlayer distances (d ) obtained from\n12\nmultiple 3D-AFM measurements at the\npristine HOPG–aqueous solution interface\nacross different electrolyte compositions\n(ion types and concentrations) and electrode\npotentials. For pure water, the 0-min data\nfrom all measured datasets (M1, M2, and M3)\nwere used (corresponding to Supplementary\nFig. 3a–c). For 0.1 M KCl, M4 (0 min, post-\nscan aging at 0, 30, 60, 90, 120 min), M5, and\nM10 (−1.78 V) were used (Supplementary\nFigs. 3d, 14a, 11a, and 13c). For 0.1 M\nK SO , datasets U4 and U6 were used (Supplementary Figs. 8 and 10). For 0.2 M K SO , datasets M8 (−1.28\n2 4 2 4\nV, −1.78 V, and post-scan aging at 20, 40, 60 min) and M9 (−1.28 V, −1.78 V) were used (Supplementary Figs.\n13a,b and 14b). For 0.01 M K SO , datasets U1, U2 (including potential scan 1: −1.6 V, −2.2 V, −2.6 V), U3\n2 4\n(including potential scan 1: −1.8 V), U5, M6, and M7 were used (Supplementary Figs. 5–7, 9, 11b,c, and 12a,b).\nReviewer comment 1.5: In the same spirit, why is the amplitude of the hydration layer oscillations\nso much smaller (1/2 o1/3) in the presence of ions versus the pure water result in Fig 2b. Likewise\nafter removal of the organic layer the amplitude of the hydration layer is reduced to ¼.\nResponse 1.5: Similar to the error analysis of d as stated in Response 1.4, we conducted\n12\nadditional thorough error analysis of the force oscillation amplitude, in order to verify whether the\nvariations of this metric are due to the inherent sample structure or external, uncontrollable factors.\nThese additional analyses are shown in the added Supplementary Fig. 16. The conclusion is that\nthese variations are due to external factors (e.g., random variations in AFM tip geometry for\ndifferent measurements).\nChanges to the manuscript:\n1) Same changes in Page 4 | Lines 28–31 and Page 4 | Lines 39–46 as stated in Response 1.4.\n2) We added Supplementary Fig. 16:\nSupplementary Fig. 16 | Force\noscillation amplitude analysis of\n3D-AFM datasets. a, A\nrepresentative 3D-AFM dataset\n(corresponding to Supplementary\nFig. 10a, −0.8 V) together with\nschematics depicting the\nextraction of the force oscillation\namplitude. b,c, Summary of\nmeasured force oscillation\namplitudes from representative\n3D-AFM datasets as a function of\nelectrode potential for (b)\npristine HOPG–electrolyte\ninterfaces (corresponding to\nSupplementary Figs. 5–10, 11a,\nand 11c) and (c) non-pristine\ninterfaces (corresponding to\nSupplementary Fig. 13) with\ndifferent electrolyte compositions\nand concentrations. The overall\nspread in force values exceeds\nany systematic dependence on\nelectrolyte concentration or ion\nidentity.\nReviewer comment 1.6: The potential dependence of the water modes in Figure 3 is very\ninteresting and the authors might to say how reproducible the effects is when you cycle the potential\nover hours (as the experiments are reported to have taken place over a span of two days in the\ncypher ES (....after looking at the supplement this question is nicely addressed please add a\ncomment to main text that points to this element of robustness for the sealed cell...this aspect is\npresently “hidden” in the supplement). How sensitive is the shift in the water modes to the identity of the supporting\nelectrolyte. The results are shown for K2SO4 but what about the results for KCl or NaCl? Is the same slope obtained in the OH-1peak position?\nResponse 1.6: We agree that argon sealing is effective in maintaining a more stable interface with\nslower ageing. However, this does not mean that experiments carried out in air are not reproducible\nor do not enable the observation of the intrinsic potential-dependence. In the original manuscript,\nat Page 8 | Lines 17–20 (Page 8 | Lines 33–36 in the revised manuscript), we mentioned that “To\nminimize the possible convolution of ageing to the potential dependence of the interfacial water\nstructure, the measurements were conducted either quickly (within ~20 minutes for each potential\nscan) to mitigate ageing or in an argon-sealed liquid cell to slow down the ageing process.” We\nbelieve this is an accurate statement regarding the effect of argon sealing. To further clarify this\neffect, we added another sentence right after the above description in the revised manuscript): “As\nshown in Supplementary Figs. 5–10, argon sealing was usually effective in preserving the pristine\ninterface for at least 30 hours.”\nRegarding the dependence of the OH stretching peak position on electrolyte identity, we agree that\nthis is an important question. Previous studies proposed some weak electrolyte-dependence of the\ninterfacial water vibrational modes (Nat. Mater. 2019, 18, 697–701; Nature 2021, 600, 81–85;\nJoule 2023, 7, 1652–1662), although a reliable conclusion cannot be reached due to the lack of\ncomprehensive control experiments and quantitative analyses. In the past four years, we have\nattempted a few SHINERS experiments on different electrolytes. However, similar to the 3D-AFM\nresults (Supplementary Fig. 15), we are not able to reach a comprehensive conclusion yet about\nthe electrolyte-dependence, due to the random fluctuations induced by external experimental\nfactors. We will continue such efforts in the future, but we believe a comprehensive study of\nelectrolyte-dependence is beyond the scope of this paper, which is mainly focused on the impact\nof environmental ageing and electrode potential on the interfacial water structure.\nChanges to the manuscript: at Page 8 | Lines 36–37, we added: “As shown in Supplementary\nFigs. 5–10, argon sealing was usually effective in preserving the pristine interface for at least 30\nhours.”\nReviewer comment 1.7: Why is the scatter band in Figure 2b for water layers KCl appear to be\nso much smaller than K2SO4.Is the difference significant, one might imagine that the halide double\nlayer might organize in a more isotropic fashion…with regard to the\nwater solvation and hydration layers?\nResponse 1.7: As explained in Responses 1.4 and 1.5, we should avoid overinterpreting\ndifferences in the observed 3D-AFM force curves, due to random fluctuations induced by AFM\ntip condition, environmental factors and instrument noise (considering the two different locations\nand four-year time span). We believe Responses 1.4 and 1.5 and the manuscript revisions specified\ntherein already addressed this concern. To further examine the direct comparison between KCl and\nK SO solutions, we have prepared Fig. R1 below using the existing data reported in the\n2 4\nmanuscript. We can see that the difference in electrolyte is not the reason why the scatter bands\nare different in Fig. 2b.\nFig. R1. Representative 3D-AFM force–distance curves showing the variation in scatter bands obtained from\nrepeated measurements on pristine HOPG–electrolyte interfaces.\nReviewer comment 1.8: This is very useful paper certain to help improve the robustness of the\napplied methods and thereby suitable for timely publication.\nResponse 1.8: We concur.\nReviewer comment 1.9: The data in the supplement clearly point to the advantages of using the\nclosed ES cell.\nResponse 1.9: We agree. Related changes in the manuscript have been specified in Response 1.6.\nReviewer comment 1.10: The idea of using a nominally outer sphere redox couple to define the\nvalue of a quasi-reference electrode is wrong. The presence of the redox couple pins the Pt\nelectrode at the E1/2 potential of the redox couple...which is different from the value of the Pt\nfloating in a K2SO4 solution. On the scale of the potential changes examined this is a minor point.\nAlthough when H2 is generated in the cell this will tend to drive the Pt reference to H+/H2\npotential that is also subject to pH shear in the non-buffered electrolyte. A better way to establish\nthe quasi-ref potential would be too compare the cyclic voltammetry in K2SO4 using a Ag/AgCl/Cl\nvs Pt and trying to minimize the extension into the HER region.\nResponse 1.10: We appreciate the reviewer’s insightful suggestions. We agree that using an outer-\nsphere redox couple to define the potential of a Pt quasi-reference electrode is not the most precise.\nAs the reviewer also noted, this is a minor point considering the large range of the potential changes\nexamined here. We also considered the suggested approach of using cyclic voltammetry in K SO\n2 4\nsolution to establish the Pt potential. However, this method is not applicable in our system because:\n• The CV of the graphite/aqueous interface is essentially featureless until the onset of hydrogen\nevolution reaction (HER) (see Supplementary Fig. 22).\n• The HER onset potential is highly variable depending on the exact surface condition and\ntherefore unreliable.\n• HER can additionally pin the Pt reference to H+/H , which is further shifted by local pH\n2\ngradients in the non-buffered electrolyte.\nTo further examine the possible errors in the Pt potential calibration, we performed additional\ncontrol experiments in which the open-circuit potential of Pt was monitored against a stable\nAg/AgCl reference electrode in 0.1 M K SO (Supplementary Fig. 30b). The obtained OCP offset\n2 4\nis 0.27 V, which is close to the 0.22 V obtained using the redox couple calibration. We therefore\nemphasize that while small variations of ~±0.1 V can be expected, they remain minor and do not\naffect the conclusions of this work.\nChanges to the manuscript:\n1) In Page 18 | Lines 29–31 of the main manuscript, we added “It is worth noting that the potential\nof a Pt quasi-reference electrode may not be exactly fixed; variations in the rough range of ±0.1 V\nmay arise but do not affect the conclusions we reached in this work.”\n2) We added Supplementary Fig. 30b:\nSupplementary Fig. 30 | Reference electrode calibration. a,\nCyclic voltammetry curves of glassy carbon electrode / 0.1 M\nK SO + 50 mM K [Fe(CN) ] + 50 mM K [Fe(CN) ] in water.\n2 4 4 6 3 6\nCounter electrode: platinum. Reference electrode (RE):\nAg/AgCl (shown in red) or platinum (shown in blue). Working\nelectrode surface area: 19.64 mm2. The potential of Pt was\nfound to be 0.22 V more positive than Ag/AgCl. b, OCP\nrecorded as a function of time for a platinum wire electrode\nversus an Ag/AgCl reference electrode in 0.1 M K SO\n2 4\naqueous solution. The average OCP value was found to be\n~0.27 V.\nReply to Reviewer 2\nReviewer comment 2.0: This study explores the complex structure of interfacial water under\nrealistic conditions by employing a combined approach of three-dimensional atomic force\nmicroscopy (3D-AFM) and interface-sensitive Raman spectroscopy (SHINERS). The focus is on\nthe graphite–water interface, which is highly relevant to applications in biology, energy\nconversion, and environmental technologies. The authors identify three distinct interfacial water\nstates: (1) a transient pristine water state near open circuit potential(OCP), characterized by\nstrong hydrogen-bond (HB) breaking; (2) a hydrocarbon-dominated state, also near OCP but more\nstable, showing weak HB breaking due to the accumulation of hydrocarbon species; and (3) a\nstable pristine water state at sufficiently negative potentials, exhibiting a broader distribution of\nHB configurations. The complementary use of 3D-AFM for spatial liquid density profiling and\nSHINERS for chemical structure identification offers in situ, high-resolution insights into the\ninterfacial water structure within ~2 nm of the surface. While the study presents a compelling\nframework, several important points need clarification before the manuscript can be recommended\nfor publication:\nResponse 2.0: We thank the reviewer for the careful evaluation and constructive comments. The\nfeedback has been highly valuable for further strengthening the manuscript. Point-by-point\nresponses are provided below.\nReviewer comment 2.1: The authors should present AFM images of the graphite surface and\nclearly indicate where the measurements were taken. Given that graphite surfaces can include step\nedges, terraces, and other defects—with known variations in adhesion properties—it is essential\nto specify whether the measurements were conducted on atomically flat terraces or at defect sites.\nRelevant prior studies, such as H. Lee et al., ACS Nano 9, 3814–3819 (2015) and Z. Ye et al., Appl.\nPhys. Lett. 106, 231603(2015), should be cited to contextualize this variability.\nResponse 2.1: We agree that the surface condition is an important factor to consider. All AFM\nstudies presented in this manuscript were performed on atomically flat terrace sites. To further\nclarify this, we have added an atomic-resolution image showing the graphitic lattice\n(Supplementary Fig. 1), where 3D-AFM measurements were subsequently carried out. The\nbehavior of interfacial structures at edge and other defect sites is beyond the scope of the present\nstudy. However, in a separate study, we have found exciting emergent effects of a series of solid–\nliquid interfaces at defect sites, which we will submit as another manuscript. We have added\nstatements in the main manuscript to clarify the measurement sites and cited the references (Refs.\n38 and 39) suggested by the reviewer.\nChanges to the manuscript:\n1) On Page 3 | Lines 30–31 & Page 4 | Lines 1–2 of the main manuscript, we have revised the\noriginal simplified statements on 3D-AFM imaging on HOPG surface to: “All 3D-AFM\nmeasurements were performed on atomically flat terrace sites of HOPG immersed in water or\naqueous solution (Supplementary Fig. 1). The HOPG surface remained stable over time and after\npotential cycles (Supplementary Fig. 2). While step edges exist on the HOPG surface38,39, those\nsites were not examined by 3D-AFM in this work.”\n2) We added Supplementary Fig. 1:\nSupplementary Fig. 1 | AFM phase image of a pristine\nHOPG surface immersed in water. All 3D-AFM\nmeasurements in this work were conducted in regions similar\nto the one depicted here. The lattice-resolution image reveals\na hexagonal lattice with a lattice constant of ~2.5 Å. The image\nwas obtained hundreds of nm away from any step edge.\nImaging was performed at Madrid, using PPP-FM cantilevers\n(Nanosensors) operated at the second eigenmode (AC,\namplitude modulation mode). Experimental parameters: free\namplitude, 200 pm; setpoint amplitude, 150 pm; resonant\nfrequency, 195.652 kHz; spring constant, 100 N m−1; quality\nfactor, 10.5.\nReviewer comment 2.2: To rule out the influence of potential adsorbates formed during\nelectrochemical measurements, the authors should provide AFM images of the graphite surface\ntaken both before and after the experiments. This would help confirm that the observed interfacial\nstructures are intrinsic rather than artifacts of surface modification.\nResponse 2.2: We have now performed the experiment suggested by the reviewer. We imaged a\ngraphite surface immersed in a 200 mM K SO solution before and after the application of −1 V\n2 4\n(vs Pt). A comparison of tapping mode AFM images before and after the application of this\npotential did not show any obvious difference (Supplementary Fig. S2).\nChanges to the manuscript:\n1) At Page 3 | Line 31 & Page 4 | Line 1 of the main manuscript, we added “The HOPG surface\nremained stable over time and after potential cycles (Supplementary Fig. 2).”\n2) We added Supplementary Fig. 2:\nSupplementary Fig. 2 | Large-\narea AFM images of HOPG\nsurface in aqueous solution.\nLocation: Madrid. Electrolyte:\n200 mM K SO in water.\n2 4\nHOPG surface topography\nmaps were measured at 0 V, (a)\nbefore and (b) after applying\n−1 V vs Pt for 2 min. The\ncross-sectional height profiles\nare shown below the\ncorresponding images,\nindicating no change of the\nstep height. Imaging mode:\nAC, amplitude modulation,\nfirst eigenmode. AFM\ncantilever: ArrowUHFAuD\n(Nanosensors). Imaging\nparameters: resonant frequency, 423.94 kHz; spring constant, 5.35 N m−1; quality factor, 4.2; free amplitude,\n3.8 nm; setpoint amplitude, 2.73 nm.\nReviewer comment 2.3: The mechanical pressure exerted by the AFM tip during 3D-AFM\nmeasurements is expected to be significantly higher than that in SHINERS. The authors should\ndiscuss whether this difference in applied pressure could influence the water structure near the\ninterface and whether it could account for any observed discrepancies between the two techniques.\nResponse 2.3: We appreciate the reviewer’s insightful comment regarding the possible effect of\ntip pressure in 3D-AFM compared to SHINERS. We address this in the following sections,\nconsidering (i) the possible deformation of the graphite substrate under the applied load, and (ii)\nthe possible influence of the AFM tip on the interfacial liquid structure.\n1. Graphite deformation under applied load:\nTo evaluate whether the mechanical load in our measurements could perturb the interfacial\nwater structure, we estimated the elastic deformation of a graphite surface under an AFM tip\nusing the Hertz contact model. The effective modulus 𝐸∗ of a silicon probe interacting with a\ngraphite substrate is given by\n1 1−𝜈(cid:2870) 1−𝜈(cid:2870)\n(cid:3047) (cid:3046)\n(cid:3404) (cid:3397)\n𝐸∗ 𝐸 𝐸\n(cid:3047) (cid:3046)\nwhere Young’s modulus 𝐸 ≈ 160 GPa and Poisson ratio 𝜈 ≈ 0.278 for silicon (J.\n(cid:3047) (cid:3047)\nMicroelectromech. Syst. 2010, 19, 229–238; Nat. Commun. 2017, 8, 1944) and 𝐸 ≈ 36 GPa\n(cid:3046)\nand 𝜈 ≈ 0.24 for graphite (Nat. Commun. 2017, 8, 1944; Phys. Rev. B 2007, 75, 153408),\n(cid:3046)\nyielding 𝐸∗ ≈ 31 GPa. Under typical 3D-AFM measurement conditions (tip radius R ≈ 5 nm,\n(cid:2870)/(cid:2871)\n(cid:2871)(cid:3007)\nmaximum load of F ≈ 500 pN), the maximum indentation depth is 𝛿 (cid:3404) (cid:4672) (cid:4673) ≈ 0.31 Å,\n(cid:2872)(cid:3006)∗√(cid:3019)\nwhich is substantially smaller than the thickness of a single interfacial water layer (≈3 Å).\nTherefore, the effect of the mechanical deformation in the 3D-AFM data is negligible.\nFurthermore, the pressure-induced substrate deformation, if any, is only expected to occur after\nthe probe penetrates through the first interfacial water layer. Thus, any elastic deformation\nwould only influence the spacing between the observed substrate surface and the first liquid\nlayer, and have no impact on the separation between the upper liquid layers. Since our results\nconcern the periodic structuring of the liquid layers, all our analyses will not change even if\nthe substrate do have some deformation when the tip reaches the very end of the approach\ncurve.\nIn a separate study, we have conducted 3D-AFM imaging of an aqueous solution at an HOPG\nstep edge site. As shown in Fig. R2 below, we observed an HOPG step height of ~3 Å and\nhydration layer spacing of ~3 Å, both consistent with standard or expected intrinsic structure\nof HOPG and water. We thus conclude that the small force used in 3D-AFM does not result in\nobservable structural distortions of the whole interface.\n[redacted]\n2. Effect of the tip on the interfacial liquid structure:\nIt is important to clarify that the oscillatory forces measured in 3D-AFM do not arise from\nmechanical compression of water molecules. Instead, those forces reflect the local\nconfigurational free energy of interfacial water, which is largely entropic in nature. Our recent\nperspective (J. Phys. Chem. C 2025, 129, 5273–5286) discussed this mechanism extensively.\nThe observed force oscillations represent probe-induced modulations of interfacial liquid\nconfigurational entropy, rather than a hydrostatic pressure. Unlike in surface force apparatus\n(SFA) measurements where two extended surfaces strongly confine the liquid, 3D-AFM\nprobes a localized volume in which water molecules remain free to diffuse and reorganize\ndynamically. Consequently, the oscillatory forces correspond directly to the intrinsic liquid\ndensity oscillation at the interface.\nIn addition, experimental evidence supports this interpretation: in Figure 9 of J. Phys. Chem.\nC 2025, 129, 5273–5286 (reproduced below), nearly identical oscillatory force curves were\nobtained using AFM tips with radii of 10 nm and 250 nm, demonstrating that the observed\nsolvation forces do not depend on mechanical indentation. Similarly, our recent work (Proc.\nNatl. Acad. Sci. U.S.A. 2025, 122, e2421635122; Fig. S5) showed analytically that only the\nterminal water molecule(s) adjacent to the tip contributes to the measured solvation force,\nwhich can be expressed as a function of the intrinsic, unperturbed local liquid density. Thus,\n3D-AFM captures the entropic reorganization of interfacial water rather than its mechanical\ncompression.\nChanges to the manuscript:\n1) At Page 2 | Lines 36–37 of the main manuscript, we added: “On the other hand, 3D-AFM\ncaptures the local liquid density30,33 without inducing observable substrate deformations\n(Supplementary Note 1).”\n2) We added Supplementary Note 1:\nReprinted with permission from J.\nR eprinted from Proc.\nNatl. Acad. Sci. U.S.A. 2025, 122, e2421635122. Copyright 2025 the Author(s)\nUsing the Hertz contact mechanics model, the deformation (indentation) of the graphite substrate\nunder our imaging conditions can be estimated. The effective elastic modulus 𝐸* for a silicon tip\nin contact with graphite is\n1 1−𝜈(cid:2870) 1−𝜈(cid:2870)\n(cid:3047) (cid:3046)\n= + ,\n𝐸* 𝐸 𝐸\n(cid:3047) (cid:3046)\nwhere Young’s modulus 𝐸 ≈ 160 GPa and Poisson ratio 𝜈 ≈ 0.278 for silicon1,2. For graphite,\n(cid:3047) (cid:3047)\nthese values are 𝐸 ≈ 36 GPa and 𝜈 ≈ 0.242,3. Inserting these values into the above formula,\n(cid:3046) (cid:3046)\nwe obtain 𝐸∗ ≈ 31 GPa. For a spherical tip of radius R under a normal load F, the indentation 𝛿\nis given by the Hertz relation:\n3𝐹 (cid:2870)/(cid:2871)\n𝛿 = (cid:3436) (cid:3440)\n4𝐸∗√𝑅\nUsing representative 3D-AFM parameters (tip radius R ≈ 5 nm, maximum load of F ≈ 500 pN),\nthe indentation depth is 𝛿 ≈ 0.31 Å. This value is substantially smaller than the interlayer\ndistance of pristine interfacial water (~3 Å).\nReviewer comment 2.4: The observed shift in interlayer spacing from ~3 Å to 4–5 Å over the\ncourse of one hour warrants further explanation. First, how is the “0-minute” time point defined?\nSince time is required for probe alignment and approach, there may be an earlier,\nunobserved state preceding the nominal start of measurement. Second, considering the layered nature of graphite,\ntip-induced deformation or water intercalation between the top and sub-surface graphene layers\nmay contribute to this evolution. The authors should clarify the physical origin of the time-\ndependent changes in interlayer spacing and discuss the potential role of tip-sample interactions\nor interfacial water penetration.\nResponse 2.4: We appreciate the reviewer’s quest for a thorough evaluation of alternative\nexplanations for the evolution of liquid layer spacing. These points are addressed below.\n1. Definition of time-zero and initial interfacial structure:\nImportantly, the ~3 Å interlayer spacing corresponds to well-established intrinsic layer spacing\nin pure water (see previous reports: Nat. Commun. 2016, 7, 12164; Nat. Commun. 2017, 8,\n2111; ACS Nano 2012, 6, 9013–9020; Proc. Natl. Acad. Sci. U.S.A. 2024, 121, e2407877121). \nThus, defining this point as “time zero” is physically justified—the system is at its intrinsic\nbaseline condition, and the precise elapsed time since sample preparation is not critical to the\ninterpretation of the subsequent evolution. We are not aware of any theoretical predictions of\nan interlayer spacing smaller than 3 Å for interfacial water, and have never observed such small\nspacings (beyond the experimental error range) at HOPG–water interface regardless of how\nfast we prepare the sample and probe. Therefore, it is safe to assume that there is no\n“unobserved state preceding the nominal start of measurement.”\nIn the actual experiments, the total duration from graphite exfoliation (to expose a pristine\nsurface) to probe alignment and initiation of 3D-AFM measurement is in the scale of 5 minutes,\nwhich is almost negligible compared to the 1–2 hours required to observe the 3 Å to 4–5 Å\nshift in liquid layer spacing. For this reason, we specified in the original manuscript that\n(originally Page 8 | Lines 8–10, now Page 8 | Lines 24–26): “State 1 was achieved by either\nlimiting the total sample preparation/air exposure time to no more than a few minutes before\nstarting the measurement, or via electrochemical cleaning after the sample was prepared (as\ndiscussed later).” The “0-minute” time point corresponds to the time when the first 3D-AFM\nforce map was acquired for the pristine HOPG–water interface.\n2. Tip-induced deformation and intercalation:\nWe have thoroughly discussed possible tip-induced deformations in Response 2.3. Briefly, our\ncalculations indicate that graphite deforms less than 0.3 Å throughout the whole 3D-AFM\nmeasurement process, which has negligible impacts on the conclusions. Our measurements at\nHOPG step edges revealed a constant step-height at ~3 Å (Fig. R2), eliminating any possibility\nof intercalation effects. Furthermore, electrochemical intercalation of ions or protons is not\nfeasible under our conditions, as the required insertion potentials (~ –2.5 to –3 V vs SHE) lie\nbeyond the range of our measurements (see results in Sci. Rep. 2016, 6, 28421; Adv. Sci. 2017,\n4, 1700146). Consistent with existing studies, in our cyclic voltammetry measurements we did\nnot detect any peaks that would be indicative of any intercalation effects (see Supplementary\nFig. 22).\nAs to the reviewer’s request that “The authors should clarify the physical origin of the time-\ndependent changes in interlayer spacing”, this was already clarified in Page 4 | Lines 2–5 of the\noriginal manuscript (Page 4 | Lines 8–10 in the revised manuscript): “The observed time-evolution\nis consistent with previous reports6,25,40. As explained before, ~3 Å corresponds to pristine\nhydration layers, while 4–5 Å is likely due to the accumulation of adventitious species whose exact\nnature remains unknown to date6,25,40.” This explanation is further corroborated by our SHINERS\nmeasurements revealing the accumulation of hydrocarbons after ageing in air (Fig. 2c) and our\ncontrol experiments where the ~3 Å spacing is preserved over 1–2 days in an electrochemical cell\nsealed in argon gas (Supplementary Figs. 5–10).\nChanges to the manuscript:\n1) The same changes specified in Response 2.3 that addressed the concern on possible substrate\ndeformation.\n2) At Page 4 | Lines 6–8 of the main manuscript, we added: “The “0 min” time point corresponds\nto the time when the first 3D-AFM data frame was acquired at the pristine HOPG–water\ninterface.”\nReply to Reviewer 3\nReviewer comment 3.0: This is an exciting fundamental study of the water/solid interface using\na combination of three-dimensional atomic force microscopy and interface-sensitive Raman\nspectroscopy. (3D-AFM and SHINERS) It is used to probe of dynamic hydrogen bonding network\nat the interface to understand the role of salt and organic species within the interfacial region\nwithin 2 nm of graphite surface. They observe two interfacial configurations, transient state\npristine water with strong hydrogen bonds, and another, with hydrocarbons dominating the\ninterface, and weak hydrogen bond breaking. As far as I can tell the procedures are reasonable,\nthough I am not an experimentalist. The insight of the observations motivates exploration of\ntheoretical and simulation models of the phenomena observed in this complex system.\nCommunication of the details and findings will benefit the community. Please publish.\nResponse 3.0: We thank the reviewer for the evaluation of our work from a theoretical perspective.\nWe agree that the publication of this work will significantly benefit the community and motivate\nfurther theoretical/computational studies.\nReply to Reviewer 4\nReviewer comment 4.0: This paper studies the interfacial water structure on the HOPG using a\ncombination of 3D-AFM and SEIRAS. While 3D-AFM has greatly advanced our understanding of\nthe liquid-solid interface by providing visualization of the interfacial molecule arrangement, it\noffers limited information on the physicochemical interactions between them. This paper provides\na comprehensive picture of the interfacial water structure on both pristine and hydrocarbon-\nadsorbed HOPG. The correlative analysis of 3D-AFM and SEIRAS bridges the gap between two\ncontrasting models of the HOPG-water interface: majorly water at the surface and majorly\nadventitious non-water molecules at the surface. They demonstrate a time-and potential-\ndependent transition between two states. In addition, they report disrupted hydrogen bonding (HB)\nat the pristine HOPG-water interface, whereas the hydrocarbon-covered surface shows less\nbroken HB. Given that the crucial roles of interfacial water in physics, biology, and especially\nchemistry as solvent, reactant, and proton donor to modulate the activation energy of chemical\nreactions, this well-written paper will be of broad interest to the readership of Nature\nCommunication. The high-quality correlative approach could serve as a valuable framework for\nfuture studies of interfacial water structures. We therefore support publication of this manuscript\nafter the authors address the following minor comments.\nResponse 4.0: We thank the reviewer for the careful evaluation of this manuscript and for the\nconstructive suggestions. We have addressed the remaining minor comments as detailed below.\nReviewer comment 4.1: The authors conclude that pristine HOPG, rather than adsorbed\nhydrophobic molecules (probably adventitious hydrocarbons), weakened the HB network of water\nmolecules leading lower HB-bonded or non-HB-bonded water molecules. Since HOPG itself is a\ncarbon-based materials, what kinds of characteristic of HOPG may contribute to this difference?\nResponse 4.1: Indeed, this is an intriguing observation emerging from our data. Both pristine\nHOPG and adventitious hydrocarbons weaken the hydrogen-bond (HB) network of interfacial\nwater, as reflected by vibrational peak shifts in the SHINERS spectra relative to bulk water.\nAdditionally, our data suggest that pristine HOPG induces a more pronounced HB disruption,\nresulting in a larger fraction of weakly H-bonded water. While an understanding of the exact origin\nof this effect will require atomistic simulations that are beyond the scope of this work, here we\npropose qualitative explanations based on our observations and prior literature.\n1. Structural rigidity/dynamics: The rigidity and planarity of the HOPG surface may limit\nthe configurational flexibility of nearby water molecules, thereby exacerbating the\ndisruption of tetrahedral water structures. In contrast, the dynamically moving, less dense\ninterfacial hydrocarbon molecules may be less effective in interrupting the HB network of\nthe surrounding water. This point was already discussed in the original manuscript\n(originally Page 7 | Lines 9–13, now Page 7 | Lines 21–25): “This reveals that the\nhydrocarbon layers are less effective in breaking HBs of nearby water compared to HOPG.\n...due to the weaker hydrophobic interaction between water molecules and the dynamically\nmoving, less dense hydrocarbon layers (aged interface) compared to the fixed, densely\npacked HOPG substrate (pristine interface).”\n2. Chemical interactions: Although both HOPG and adventitious hydrocarbons are carbon-\nbased, their physicochemical properties differ substantially. HOPG presents an atomically\nflat, extended sp2 carbon lattice with delocalized π electrons, offering a non-polar, highly\nordered, and relatively inert surface. In contrast, hydrocarbons, while generally considered\nnonpolar, contain C–H bonds which possess weak dipole moments due to the\nelectronegativity difference between carbon and hydrogen. This subtle polarity can support\ndipole–dipole or dipole–induced dipole interactions with nearby water molecules,\npotentially mitigating HB disruption (Science 2021, 374, 1366–1370; J. Phys. Chem. B\n2023, 127, 3798–3805; J. Phys. Chem. B 2002, 106, 2047–2053). We have now added this\ndiscussion in the revised manuscript.\nChanges to the manuscript: At Page 7 | Lines 25–28 of the main manuscript, we added “In\naddition, adventitious hydrocarbons contain C–H bonds with small dipole moments, which can\nenable weak dipolar interactions with the surrounding water62, thus mitigating HB-disruption\neffects.”\nReviewer comment 4.2: The authors should discuss potential zero charge (PZC) of the HOPG?\nThe direction and the strength of the interfacial electric field should be discussed with reference\nto the PZC (rational potential scale).\nResponse 4.2: We thank the reviewer for this thoughtful suggestion. To address this point, we\nperformed electrochemical impedance spectroscopy (EIS) measurements on HOPG in 0.1 M\nK SO (see the added Supplementary Fig. 31). EIS is the most common method for estimating the\n2 4\npotential of zero charge (PZC), with the capacitance minimum typically taken as the potential of\nzero charge.\nOur measurements show an expected minimum in the capacitance vs potential profile, consistent\nwith prior studies of graphite in aqueous solutions (J. Electrochem. Soc. 1971, 118, 711; J. Phys.\nChem.1985, 89, 4249–4251; Langmuir 2016, 32, 11448–11455). However, the assignment of this\nminimum to a well-defined PZC is actually not straightforward.\nEarly studies by Randin and Yeager (J. Electrochem. Soc. 1971, 118, 711; J. Electroanal. Chem.\n1972, 36, 257–276) and Gerischer (J. Phys. Chem. 1985, 89, 4249–4251) highlighted that the\ngraphite electrodes exhibit an unusually low capacitance minimum (~3–4 μF cm−2). They\nattributed this to the limited electronic density of states at the Fermi level. They argued that this\nminimum corresponds to a “potential of zero space charge (PZSC)” rather than the true\nthermodynamic PZC.\nFurthermore, PZC is expected to vary depending on the exact interfacial state (pristine vs.\nnon-pristine), as the accumulation of adventitious hydrocarbons at the interface (displacing interfacial\nwater) can easily alter the PZC. A definitive determination of the PZC for these distinct surface\nstates would require dedicated EIS measurements in conjunction with 3D-AFM and SHINERS,\nwhich lies beyond the present scope.\nDue to the above uncertainties, we refrain from assigning the observed capacitance minimum as a\nuniversal PZC at HOPG–aqueous solution interfaces.\nChanges to the manuscript:\n1) We added a paragraph in the main manuscript (Page 18 | Lines 33–37): “Additionally,\nelectrochemical impedance spectroscopy (EIS) measurements of HOPG in 0.1 M K SO solution\n2 4\nrevealed a minimum differential capacitance at –0.6 V vs Pt (–0.38 V vs Ag/AgCl) (Supplementary\nFig. 31). While this value may be close to the potential of zero charge (PZC), the exact PZC value\nmay change at different interfacial states, depending on the presence/absence of adventitious\nhydrocarbons”\n2) We added Supplementary Fig. 31 to the Supplementary Information:\nSupplementary Fig. 31 |\nElectrochemical impedance\nspectroscopy (EIS)\nmeasurements and\ncapacitance analysis.\nElectrode: HOPG.\nElectrolyte: 0.1 M K SO\n2 4\naqueous solution. a, EIS\nspectra at a series of\npotentials vs Pt, plotted in\ncomplex capacitance plane.\nMeasurements were\nperformed using a Biologic\npotentiostat over the\nfrequency range of 1 Hz to 1\nMHz with an AC perturbation amplitude of 25 mV. The electrode surface area was 0.64 cm2. Data in the\nrange\nof 316 Hz to 1 MHz were used for fitting. Scattered dots correspond to the experimental data and lines are series\nRC circuit fits. b, Capacitance as a function of potential, extracted from RC circuit fits in (a). The capacitance\nvs potential curve reveals a minimum at –0.6 V vs Pt (–0.38 V vs Ag/AgCl).\nReviewer comment 4.3: I recommend against defining ‘0-0.4 V as OCP’. This is an enormously\nbroad potential range and should not collectively be called OCP.\nResponse 4.3: We appreciate the reviewer’s rigorous examination of the nomenclature. Our\nintention was not to redefine OCP, but rather to highlight that across this range no differences can\nbe observed in either 3D-AFM or SHINERS results. Moreover, in the absence of reversible redox\nreactions to pin it, the OCP of the graphite–aqueous solution interface is not a fixed value. It varies\nwith electrolyte composition and the surface condition of HOPG, and thus we cannot assign a\nsingle definitive potential to the large amounts of samples we measured. Nevertheless, to avoid\npotential confusions, we have changed the phrases used in the corresponding sentence as described\nbelow.\nChanges to the manuscript: At Page 3 | Line 28 of the main manuscript, we changed “are all\nregarded as OCP” to “can be regarded as at or near OCP”.\nReviewer comment 4.4: The SHINERS particles will form their own double layer which will\npotentially be influenced by changes in the charge distribution imparted by the application of\ndifferent potentials to the working electrode. The authors point to other references that discuss the\nnon-interaction of these particles. However, these discussions need to be present in the current\nmanuscript to make the data credible. Thus, relevant discussion needs to be added.\nResponse 4.4: We agree with the reviewer that this is a potential concern that is worth clarification.\nDespite the inertness of the silica shell, the possibility of double-layer effects from the SHINERS\nparticles must be considered. In fact, this point was already addressed in the original manuscript\n(originally Page 7 | Lines 15–25 and Supplementary Fig. 17; now Page 7 | Lines 30–41 and\nSupplementary Fig. 21). Specifically, we wrote:\n“In SHINERS measurements, the active interfacial liquid regions contributing to the signal are\nsandwiched between the HOPG surface and SiO (shell of the Au/SiO particles) (Fig. 1d). To rule\n2 2\nout the possible role of silica in producing the emergent interfacial ν peaks (OH-3 to OH-5), we\nOH\ndesigned and fabricated a control sample, Au film/SiO /electrolyte/SiO /Au nanoparticles. As\n2 2\nshown in Supplementary Fig. 17, the silica layer is ~3 nm thick at the active gap region. This\nsample serves as a well-defined control system that enables Raman enhancement, is graphite-free,\nand ensures SiO is the dominant surface composition in contact with the probed volume of\n2\naqueous solution. SHINERS spectra of this control sample at multiple different areas and varying\nelectrode potentials did not exhibit OH-3 to OH-5 peaks (Supplementary Fig. 17), distinct from\nthe results obtained for HOPG/solution interfaces. These data prove that the silica shell layer does\nnot directly contribute to the SHINERS peaks of the HOPG–water interface.”\nSince our carefully designed control samples described above also contain the electrical double\nlayers formed on the silica shell, we believe the existing control experiments also verified that the\nsilica shell’s double layers are not a concern. As such, we have revised the last sentence in the\nabove-mentioned paragraph to clarify this point.\nChanges to the manuscript: Page 7 | Lines 39–41: “These data prove that neither the silica shell\nlayer nor the electrical double layer formed on the shell contributed measurably to the SHINERS\npeaks of the HOPG–water interface.”\nReviewer comment 4.5: The authors mention that minimal changes are observed with the\npresence of ions but Figure 2b shows considerable changes in the force curves. These changes\nneed to be more clearly discussed and elaborated.\nResponse 4.5: We thank the reviewer for this important observation. Our statement regarding\n“minimal changes” was specifically referring to the interlayer spacing d , which remains\n12\nconsistent across different electrolytes. We agree that Figure 2b displays noticeable variations in\nthe force magnitudes and overall curve profiles. However, these variations are not systematically\ncorrelated with ion presence or electrolyte identity. Instead, they primarily arise from variations in\nexternal factors such as AFM tip condition, environmental factors and instrument noise\n(considering the two different locations and four-year time span). This interpretation is supported\nby our extensive analysis of the large amounts of data sets together (including measurements from\nboth Urbana and Madrid), which shows that the spread in force values is substantially larger than\nany systematic dependence on electrolyte concentration or ion identity. We have added extensive\nerror analyses and related discussions in the revised manuscript.\nChanges to the manuscript:\n1) The original statement in the main manuscript Page 4 | Lines 22–23, “This interpretation is\nconsistent with prior reports that showed the salt-independence of hydration layer spacings in\ndilute solutions (<1 M)”, is now changed to (Page 4 | Lines 28–31) “This interpretation is\nconsistent with prior reports suggesting that hydration layer spacings are largely insensitive to the\nidentity and concentration of the supporting electrolyte in dilute solutions (<1 M), since the\nnumber density of water is much higher than that of the ionic species in these solutions”.\n2) We added a new paragraph in the main manuscript to further clarify the error analysis and\npotential origins of experimental errors (Page 4 | Lines 39–46): “Additionally, care must be taken\nto avoid overinterpreting differences in the observed 3D-AFM force curves. Random fluctuations,\ndue to variations in AFM tip condition, environmental factors and instrument noise (considering\nthe two different locations and four-year time span), can result in various error ranges in the\nexperimental observables. For example, average d values within 2.8–3.3 Å were observed for\n12\npristine HOPG–aqueous solution interfaces with different ionic species and concentrations\n(Supplementary Fig. 15). Force oscillation amplitudes, on the other hand, exhibits larger\nfluctuations between 0.01–0.1 nN (Supplementary Fig. 16). These variations should not be directly\nattributed to differences in the electrolytes or ageing/cleanliness conditions.”\n3) We added Supplementary Fig. 15:\nSupplementary Fig. 15 | Summary of\ninterlayer distances (d ) obtained from\n12\nmultiple 3D-AFM measurements at the\npristine HOPG–aqueous solution interface\nacross different electrolyte compositions\n(ion types and concentrations) and electrode\npotentials. For pure water, the 0-min data\nfrom all measured datasets (M1, M2, and M3)\nwere used (corresponding to Supplementary\nFig. 3a–c). For 0.1 M KCl, M4 (0 min, post-\nscan aging at 0, 30, 60, 90, 120 min), M5, and\nM10 (−1.78 V) were used (Supplementary\nFigs. 3d, 14a, 11a, and 13c). For 0.1 M\nK SO , datasets U4 and U6 were used (Supplementary Figs. 8 and 10). For 0.2 M K SO , datasets M8 (−1.28\n2 4 2 4\nV, −1.78 V, and post-scan aging at 20, 40, 60 min) and M9 (−1.28 V, −1.78 V) were used (Supplementary Figs.\n13a,b and 14b). For 0.01 M K SO , datasets U1, U2 (including potential scan 1: −1.6 V, −2.2 V, −2.6 V), U3\n2 4\n(including potential scan 1: −1.8 V), U5, M6, and M7 were used (Supplementary Figs. 5–7, 9, 11b,c, and 12a,b).\n4) We added Supplementary Fig. 16:\nSupplementary Fig. 16 | Force\noscillation amplitude analysis of\n3D-AFM datasets. a, A\nrepresentative 3D-AFM dataset\n(corresponding to Supplementary\nFig. 10a, −0.8 V) together with\nschematics depicting the\nextraction of the force oscillation\namplitude. b,c, Summary of\nmeasured force oscillation\namplitudes from representative\n3D-AFM datasets as a function of\nelectrode potential for (b)\npristine HOPG–electrolyte\ninterfaces (corresponding to\nSupplementary Figs. 5–10, 11a,\nand 11c) and (c) non-pristine\ninterfaces (corresponding to\nSupplementary Fig. 13) with\ndifferent electrolyte compositions\nand concentrations. The overall\nspread in force values exceeds\nany systematic dependence on\nelectrolyte concentration or ion\nidentity.\nReviewer comment 4.6: How do the authors distinguish hydrocarbon adsorption from potential\nslow formation of oxygen groups on the HOPG surface that could also lead to structural changes?\nIt would have been nice to show measurements where organics are purposefully added to the\nelectrolyte to corroborate the claim that the changes in force curves occur because of organics\nadsorption. I realize, however, that this may be beyond the scope of the study.\nResponse 4.6: We thank the reviewer for this thoughtful comment. The distinction between\nhydrocarbon adsorption and the slow formation of oxygen-containing functional groups on the\nHOPG surface is indeed important, as both could in principle lead to changes in interfacial\nstructure.\nImportantly, the entire potential range we explored in this work lies within a regime where HOPG\nis known to remain stable (see Supplementary Fig. 22 as proof). In this range, oxygen groups are\nnot expected to form (J. Power Sources 2008, 185, 740–746; Anal. Chem. 1993, 65, 1378–1389;\nSci. Rep. 2016, 6, 22056).\nFurther, our conclusion on interfacial hydrocarbon accumulation is supported by multiple existing\npieces of evidence:\n1. Spectroscopic evidence: Parallel SHINERS measurements, as discussed in the\nmanuscript, revealed the emergence of C–H stretching modes and other hydrocarbon-\nrelated spectral features, consistent with organics rather than covalently bound oxygen\ngroups.\n2. Oscillatory force profile: At the non-pristine interface, we observed periodic damped\noscillations in the force curves, characteristic of layered liquid structures. In contrast,\noxygen groups covalently bound to the HOPG surface would be expected to form a single\nadsorbed solid layer, which cannot produce force oscillations with multiple peaks.\n3. Control experiments with organic solvents: Previous studies on pure solvent (hexane) at\nHOPG surface (Nanoscale 2021, 13, 5275–5283) indeed showed force oscillations with an\ninterlayer spacing of ~4–5 Å, closely matching that of the interfacial State 2 reported in\nthis work.\nTaken together, these results strongly support our conclusion that the observed interfacial evolution\noriginates from hydrocarbon accumulation rather than slow oxidative modification of the HOPG\nsurface.\nChanges to the manuscript: All of the above points have been discussed in multiple places\nthroughout the manuscript. To further clarify that HOPG is stable in the measured potential range,\nwe added one sentence in the caption of Supplementary Fig. 22: “No other redox reactions are\nobserved.”\nReply to Reviewer 5\nReviewer comment 5.0: I co-reviewed this manuscript with one of the reviewers who provided\nthe listed reports. This is part of the Nature Communications initiative to facilitate training in peer\nreview and to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse 5.0: We would like to thank the reviewer for helping with this process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a nice job addressing my questions. The paper is suitable for publication.", "reviewer_2": "I have carefully reviewed the revised manuscript and the authors’ response letter. The authors have addressed my\ncomments and concerns appropriately, and the manuscript has been significantly improved. I therefore recommend that the\npaper be accepted in its current form.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter