Hierarchical Assembly of Hemin-Peptide Catalytic Systems on Graphite Surfaces

Abstract

The formation of molecular hybrid systems with cofactors and peptides on graphite electrodes has recently been demonstrated. The design of peptide sequences is crucial for forming robust catalytic molecular systems on electrodes. However, the relationship between peptide sequences, molecular structure, and catalytic performance has not been fully explored. In this study, we employed peptides with simple dipeptide repeats, which effectively immobilize hemin, to construct a stable catalytic system and investigated the molecular basis of their self-assembly and catalytic activity by varying the sequence. Among peptides containing the dipeptide sequences (YH, VH, and LH), YH demonstrated the most efficient immobilization of hemin, which is catalytically active in electrochemical reactions. Using advanced molecular visualization techniques, specifically frequency modulation atomic force microscopy (FM-AFM), we characterized the well-ordered structures of these peptides on graphite electrodes, revealing their molecular-scale organization. Our findings in electrochemical characterizations include a quantitative evaluation of the surface density of hemin immobilized by self-assembled peptides and the catalytic activity of the peptide-hemin hybrid system under electrochemical conditions in the presence of H₂O₂. The strong peptide–peptide and peptide-hemin interactions, facilitated by π–π interactions of tyrosine residues, contribute to the system’s stability and efficiency. The dipeptide repeats serve as a useful platform to investigate the role of important amino acids, beyond histidine, in stably immobilizing cofactors. These results highlight the potential for developing durable and efficient catalytic interfaces in electrochemical applications.

Introduction

The development of artificial enzymes with cost-effective and scalable production methods is advancing rapidly,¹⁻³ particularly due to their potential applications in biosensing^4,5 and chemical synthesis.⁶ A promising strategy for creating these enzymes involves the self-assembly of peptides, which can be engineered to form well-defined structures and interact with cofactors, thereby mimicking the catalytic properties of natural enzymes. When combined with electrochemistry, bioelectrocatalysis has expanded scope, including the immobilization of natural enzymes on electrodes using diphenylalanine peptides⁷⁻⁹ and their integration with cofactors.¹⁰ Several studies have reported this potential, especially with peptides that self-assemble into two-dimensional crystals of monomolecular thickness, offering a pathway to highly organized peptide structures with enhanced catalytic activity.¹¹

In our previous study,¹¹ we demonstrated that hemin, when immobilized on self-assembled peptides on graphite surfaces, can act as a catalyst to reduce H₂O₂, emulating the enzymatic function of horseradish peroxidase (HRP). This peptide sequence design approach shows significant promise for creating bespoke enzymes tailored to specific chemical reactions through the strategic selection of cofactors.

Despite the potential of peptide-based catalysts, a key challenge remains: the rules governing peptide design are not yet fully understood. A particularly critical gap is the limited knowledge of the intrinsic structures of self-assembled peptides in liquid environments and their corresponding electrochemical catalytic activity. This gap is further complicated by the interaction between cofactor hemins and peptides, which can significantly influence catalytic performance. No research, to our knowledge, has successfully created nanostructures of redox-active species on electrodes while controlling their orientation.

To address this challenge, our study investigates the relationship between peptide sequence, self-assembly, and catalytic activity with hemins. We focused on a simple amino acid sequence, XH, where X represents various amino acids, to explore how these sequences influence peptide structure and function on surfaces.¹² Histidine (H) was chosen for its ability to stabilize peptide structures and bind metal ions or cofactors, making it an ideal candidate for hybrid peptide-cofactor systems.

By varying the amino acid at position X, we controlled the self-assembly of peptides on surfaces and examined their interaction with hemin. Frequency-modulation atomic force microscopy (FM-AFM) was employed to obtain molecular scale images of these peptides in ordered structures under liquid environment, while electrochemical cyclic voltammetry was used to assess their ability to immobilize hemins and catalytic activity. Our results revealed that (YH)₄ peptides provided the most effective immobilization of hemin and demonstrated remarkable catalytic activity in electrochemical reactions. These findings offer valuable insights into the design of efficient peptide-based catalysts.

Results and Discussion

A previous study revealed that histidine rich peptides (XH)₄ form ordered structures on graphite surfaces.¹² As shown in Figure 1a, each peptide contains eight amino acids, and the amino acid X in the sequence includes three hydrophobic amino acids such as Y, L, and V. The side chains of these amino acids play an important role for their self-assembly characteristics and stability, and the resulting self-assembled peptide molecular architecture reflects their amino acid sequences.

Figure 1.

(a) Amino acid sequences of peptides, (b) a schematic showing the assembly of peptides and hemins on a graphite surface, (c) a schematic showing the catalytic reaction mechanism of this system.

Based on this finding, it is expected that the X is also essential in the interaction with hemins. In this work, we utilized these peptides as molecular scaffolds to immobilize hemin cofactors. The proposed mechanism involves the peptides first assembling into ordered structures on the graphite surface from the solution. Subsequently, cofactor hemins bind to the top of ordered peptides (Figure 1b). Hemins then act as catalytic centers to reduce hydrogen peroxides under an applied electrochemical potential (Figure 1c).

Recently, FM-AFM has demonstrated its ability to visualize the ordered structures of self-assembled peptides on graphite and other two-dimensional materials.¹⁷⁻²⁰ In this study, we used in situ FM-AFM to visualize the molecular units of the ordered peptide structures. Figure 2 shows high-resolution in situ images of peptides on graphite surfaces: (YH)₄, (VH)₄, and (LH)₄. All images exhibit periodic structures of peptides on the surface, where peptides align in rows with a unique direction. These peptides are highly ordered, almost like two-dimensional crystals. Interestingly, each peptide organization has specific surface morphology, including degrees of order, periodicity, and unit cell size, despite all peptides having the same amino acid sequence length.

Figure 2.

(a) In situ FM-AFM images of (YH)₄ peptides observed under DI water. (b–d) in situ FM-AFM images showing the unit cells of each peptide: (b) (YH)₄, (c) (VH)₄, and (d) (LH)₄. These images were taken under peptide solution, where the concentrations for each peptide were 1.5, 1.0, and 1.0 μM, respectively.

The unit cell of (YH)₄ had the dimension of 3.0 nm × 0.9 nm (Figures S2 and S3). The periodicity along the nanowire direction was 0.9 nm, which was larger than the typical intermolecular distance between peptides forming a β sheet structure.²¹ The distance perpendicular to the nanowire was 3.0 nm, which aligned well with the straight length of peptides with eight amino-acid long sequences corresponding to 2.8 nm. It supports that these peptides form a β sheet structure on the surface and the peptides are straight in their conformation. Furthermore, the unit cell of (VH)₄ was 2.9 nm × 0.5 nm, while the unit cell of (LH)₄ was 3.1 × 1.2 nm. The shorter distance between peptides in the (VH)₄ unit cell, particularly the 0.5 nm spacing, was half the spacing observed in the other two peptides, which had approximately 1 nm spacing. This suggests that dimerization may be occurring in (YH)₄ and (LH)₄. The same discussion was reported in a recent work visualizing self-assembled dipeptide repeat peptides on graphite or MoS₂.¹⁸

Notably, (YH)₄ exhibited the highest order among them, characterized by the most uniform structure, minimal disturbance in periodicity over a wide range, and stability against concentration changes (Figure S4). In contrast, (LH)₄ and (VH)₄ displayed more irregularities in the spacing between peptide rows, and they also showed changes in unit cell size in tandem with concentration (Figures S4 and S5). The distinction in these observations suggests that the individual (YH)₄ peptide has the most robust conformation in the crystalline form.

Next, we explored the ability of these ordered peptides to immobilize hemins on their surfaces. First, the peptides self-assembled on graphite surfaces by applying a droplet of 1.0-μM peptide aqueous solution for 1 h. Subsequently, this solution was replaced with a 1-μM hemin solution. These specific conditions were selected to achieve partially covered surface by hemin, thereby enabling the visualization of individual hemin molecules binding to the surface over time.

The surface morphology of each peptide was observed via ex situ and in situ AFM under both dry and wet conditions, respectively. The resulting ex-situ AFM images demonstrated that all the (XH)₄ peptides effectively immobilized hemins on the surface (Figures S6, S7 and Table S1). In these ex situ AFM images, particle-like features appeared on the peptide assemblies, with heights ranging from 0.5 to 2 nm, presumed to be immobilized hemin molecules. A height of around 0.5 nm indicates hemins in a lying flat (parallel) orientation, while a height of around 1 nm suggests a standing up (perpendicular) orientation. The diversity in hemin heights may also reflect aggregation, likely induced by the dehydration process when the samples were dried. Additionally, we evaluated the binding affinity of porphyrin molecules that do not contain Fe (Figure S8). The AFM images show no significant changes in the surface morphology after adding porphyrin to the peptide-functionalized graphite surfaces. This result suggests that the Fe atom in hemin is critical for its interaction with peptides, and that the binding is not solely driven by π–π stacking interactions.

To further investigate the structures of immobilized hemin molecules under aqueous conditions, we performed in situ high-resolution FM-AFM. First, (YH)₄ peptides were absorbed onto HOPG for 10 min. The surface was then rinsed with water to remove any unbound peptides. After this, the sample was incubated for 30 min before injecting 1 μM solution of hemin. Figure 3a shows FM-AFM images captured at a hemin solution concentration of 1 μM. The immobilized hemins formed wire-like patterns. The bright spots were regularly aligned along to the self-assembled peptide rows. Further magnification revealed that these hemins were laid out in straight lines, with bright spots immobilized directly on top of the peptide rows (Figure 3b,c).

Figure 3.

(a–c) In situ AFM images showing the immobilization of hemin on self-assembled (YH)₄ peptides. (d) HS-AFM image of hemin-diffusion on a self-assembled (YH)₄ peptide, and (e) formation of hemin wires along the peptide wires.

We employed high-speed AFM (HS-AFM) to visualize the time-lapse of hemin immobilization on peptide rows. Two distinct features were observed: stationary hemin wires formed along the peptide rows, and small hemin aggregates moved in a one-dimensional manner along the peptide rows (Figure 3d). Occasionally, these aggregates transitioned to adjacent rows (see Supporting Information, Movie S1). The speed of movement varied, suggesting that the aggregates hop along the peptide row. Furthermore, the extension of the hemin wire was also observed (Figure 3e). The heights of hemins were measured to be approximately 1.0–1.5 nm, indicating that the stationary hemin is likely immobilized in an upright orientation with potential π-stacking interactions between the hemin molecules (Figures S9 and S10).

These observations suggest that self-assembled peptides can serve as a template for the immobilization of hemins, followed by hemin diffusion along the peptide rows via continuous interactions with the peptides.

The immobilized hemins on the self-assembled peptides were electrochemically characterized by cyclic voltammetry (CV). The measurements were performed using a three-electrode system, with a modified HOPG electrode with hemins and peptides as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl electrode as the reference. Three mL of 100-mM PB solution (pH 7) was placed in a handmade electrochemical cell. Prior to the measurement, we purged the solution with nitrogen gas for 20 min to replace dissolved oxygen. Nitrogen gas was continuously flowed over the solution during the measurements.

Figure 4a shows cyclic voltammograms for both the unmodified HOPG electrode and the (YH)₄ peptide-hemin-modified HOPG electrode. For the HOPG alone, only the oxidation–reduction peaks of HOPG by itself were observed around E = 0.17 V and −0.08 V, which is consistent with previous observation.²² In contrast, when the HOPG was modified with hemins and peptides, additional oxidation–reduction peaks appeared at −0.35 and −0.2 V. These peaks are attributed to Fe²⁺ ⇆ Fe³⁺ at −0.35 V²³ and oxygen reduction at around −0.2 V.²⁴ Specifically, the catalytic reduction of dissolved oxygen by hemin means that dissolved oxygen is reduced to water.

Figure 4.

Cyclic voltammetry of hemin-peptide electrodes. (a) Cyclic voltammogram of (YH)₄-hemin and bare graphite electrodes. (b) CV of (YH)₄ with various scan rate, and (c) a plot showing the peak current density depending on the scan rate. (d) Hemin concentration-dependent CV of (YH)₄-hemin on graphite, (e) a plot showing cathodic and anodic peak current density depending on the concentration of hemin.

The hemin-binding capacity of the peptides on the electrode was further studied by cyclic voltammetry with varying scan rates (0.01–0.5 V/s). The electrodes were prepared by coating HOPG with self-assembled peptides, followed by incubating with 50-μM hemin solution and subsequently washing the electrode. The obtained cyclic voltammetry results are shown in Figure 4b. The surface density of hemin molecules was quantitatively estimated from the results of CV measurements, according to eq 1.²⁵

1

Here, v is the scan rate of the electrode potential, n represents the number of electrons in the reaction, F is Faraday’s constant, R is the gas constant, T is the temperature, A is the electrode area, and Γ is the surface density of the reaction points (hemins). We performed CV for each peptide at different scan rates (Figure 4c). The obtained results revealed that the peak current I_p was proportional to v for all peptides. The slope of each line indicates the difference in the surface density Γ. According to this, the peptides with higher amounts of immobilized hemin were, in order, (YH)₄, (VH)₄, and (LH)₄.

Examining the detailed differences, the amounts of hemin immobilized by the peptides were estimated to be 9.8 × 10^–11 mol/cm² for (YH)₄, 7.7 × 10^–11 mol/cm² for (VH)₄, and 6.7 × 10^–11 mol/cm² for (LH)₄. In a previous work,²⁶ the amount of hemin directly immobilized on HOPG has been experimentally determined to be 1.1 × 10^–10 mol/cm², where hemins formed a monolayer on the surface in a close-packed manner. Comparing this with values derived in this work, the surface density of hemins is similar to the amounts in the previous work, suggesting that hemins are almost close-packed and uniformly immobilized on the peptide scaffold.

The surface density of hemins was highest for (YH)₄, which has a tyrosine residue that interacts with the porphyrin ring through π–π interactions. This suggests that, among these three peptides, the X residue plays a significant role to interact with the porphyrin ring. Next, the same trend can be observed when comparing (VH)₄ and (LH)₄, which have lower surface hemin density than (YH)₄. Previous work¹² suggested that the surface modification of the HOPG electrode by (VH)₄ increased the surface hydrophobicity relative to functionalization with (LH)₄. This surface hydrophobicity might cause the difference in the surface hemin density in (LH)₄ and (VH)₄.

Next, we performed CV at samples prepared with various hemin concentrations, ranging from 20 nM to 50 μM, to roughly derive the binding constant of hemins to the peptides and evaluate the concentration required to fully cover the surface. The CV experiments were conducted using a peptide-modified HOPG electrode prepared with a 2-μM peptide solution. Hemin solutions of the desired concentration were applied to the electrode for 10 min, followed by rinsing with 10 mM PB prior to each CV measurement.

The CV results showed a drastic change in the peak current density at −0.35 V depending on the hemin concentration (Figure 4d). For analysis, we plotted the cathodic and anodic current density at −0.35 V against the logarithm of the hemin concentration (Figure 4e). The increase in cathodic peak current varied among the peptides: while (YH)₄ exhibited a linear increase on a semilogarithmic plot, (VH)₄ and (LH)₄ showed no significant increase in the peak current below a certain hemin concentration threshold. After surpassing this threshold, the peak current increased followed by a saturation. Specifically, (VH)₄ demonstrated an increase between 500 nM and 5 μM, while (LH)₄ showed an increase between 500 nM and 2.5 μM.

The catalytic activity of the hemin-peptide electrodes was evaluated using the reduction of H₂O₂. Before conducting the experiments, hemin molecules were immobilized on peptide-immobilized HOPG electrodes by applying a 50-μM aqueous solution of hemin with NaOH, as described in the Methods and Experimental Section. The corresponding reaction equation is depicted in Figure 1c. Figure 5a–c shows the voltammograms obtained at various H₂O₂ concentrations for each peptide. All voltammograms were recorded during the fifth cycle of the CV measurement after achieving equilibrium. As the H₂O₂ concentration increases, the reduction current density at both −0.35 and −0.80 V shows a respective increase. Figure 5d–f illustrates the variation in the cathodic current at −0.80 V for each peptide as a function of the H₂O₂ concentration.

Figure 5.

Cyclic voltammetry (CV) results for hemin-peptide electrodes at varying H₂O₂ concentrations. (a–c) CV curves for (YH)₄, (VH)₄, and (LH)₄, respectively. (d–f) Current density at −0.8 V as a function of H₂O₂ concentration for each peptide, with fitting curves shown as red solid lines. I_max and K_m represent the maximal current density and Michaelis–Menten constant, respectively. R² indicates the coefficient of determination for each fit.

The reduction current at −0.2 to −0.8 V originated from the associative reduction of H₂O₂, facilitated by the catalytic activity of the hemin molecules on the electrode surface.²⁷⁻²⁹ The corresponding reaction equation can be expressed as

The currents at −0.8 V increase monotonically with increasing H₂O₂ concentrations. Each peptide exhibits a distinct trend in the current depending on the concentration of H₂O₂. (YH)₄ shows a gradual increase in current density, achieving the highest value among the tested peptides (Figure 5d). Both (VH)₄ and (LH)₄ display an initial increase in current, followed by saturation of the reaction current at higher H₂O₂ concentrations, with the maximal current density being lower than that of (YH)₄.

The Michaelis–Menten model fitting highlights the differences among the peptides. The solid curves in the plots represent the fitting results, which align well with the experimental data (Figure 5d–f). The Michaelis–Menten constants (K_m) and the maximal current densities (I_max) derived from the fitting are summarized in Figure 5. The maximal currents follow the order of (VH)₄ < (LH)₄ < (YH)₄. The K_m values show the same trend, with the K_m of (YH)₄ being 2.5 mM, 2 orders of magnitude larger than that of (VH)₄ and (LH)₄.

The high catalytic activity of hemins on (YH)₄ can be attributed to the specific characteristics of the peptide, particularly its constituent amino acids: tyrosine (Y), valine (V), and leucine (L). Tyrosine residues promote π–π interactions, while valine and leucine contribute hydrophobic properties. Table 1 summarizes the key parameters estimated. The Grand Average of Hydrophobicity (GRAVY) serves as a useful indicator for comparing peptide hydrophobicity, where negative values indicate hydrophilicity and positive values indicate hydrophobicity. According to these values, (YH)₄ is the most hydrophilic peptide. The surface density of immobilized hemins, derived from the CV measurements, reveals that (YH)₄ holds the highest number of hemins, though the differences among the peptides are within the same order of magnitude, indicating no significant variation in their capacity to immobilize hemins. It was also confirmed by X-ray photoelectron spectroscopy (Supporting Information, Figure S11).

Table 1. Number Ratio of the Hemin and Peptide on the Surface.

	hemin density × 10–11 (mol/cm2)	row distance (nm)	interpeptide distance (nm)	hydropathicity (GRAVY)
(YH)4	9.81	3.0	0.90	–2.25
(VH)4	7.67	2.9	0.51	0.500
(LH)4	6.69	3.2	1.2	0.300

On the other hand, the unit cell structure of each peptide, as revealed by FM-AFM, exhibits distinct features in terms of their interpeptide distances. These peptides are expected to adopt a β-sheet structure, forming rows on the surface. While the distances between the rows show no significant differences, the interpeptide distance in (VH)₄ peptides is approximately half that of (YH)₄ and (LH)₄. This discrepancy suggests that (YH)₄ and (LH)₄ form peptide dimers within the unit cell. The double interpeptide distance has been observed in a previous report.¹⁸ The dimers may contribute to form unique sites for hemin binding. Moreover, (YH)₄ maintains a constant unit cell structure over a range of peptide concentrations, further reinforcing its stability. In contrast, (LH)₄ and (VH)₄ exhibit changes in their unit cell structures when self-assembled at varying concentrations, indicating a degree of flexibility in their configurations. Given that the surface density of immobilized hemin molecules is similar across all peptides, the maximal current densities suggest that (YH)₄ provides the most stable scaffold for hemin binding and supports the highest turnover rate for the H₂O₂ reduction process. Notably, the AFM study confirmed that the surface morphology of the peptides remained unaffected by 1 mM H₂O₂ (Figure S12). These results reveal the importance of X amino acids in creating a stable hemin-peptide hybrid system for the H₂O₂ reduction reaction. Our findings shed light on establishing a design rules governing hydrophobic residue interacting at the two-dimensional (2D)-material surface. This knowledge can aid in developing de novo peptides that enable the formation of hybrid molecular interfaces on electrochemical electrodes through self-assembly.

Conclusions

This study explores the development of molecular hybrid systems using dipeptide repeat sequences to immobilize hemin on graphite electrodes, resulting in stable and efficient catalytic interfaces. Among the peptides tested, the YH sequence demonstrated the most effective hemin immobilization and catalytic activity in electrochemical reactions. Through frequency modulation atomic force microscopy, the well-ordered molecular structures of the peptides were visualized, offering insights into their self-assembly processes. The results emphasize the significance of strong peptide–peptide and peptide-hemin interactions, particularly the π–π interactions involving tyrosine residues, in stabilizing the system. This research highlights the potential of simple peptide designs to create artificial enzymes with robust and durable catalytic interfaces for electrochemical applications. Furthermore, the peptides’ ability to self-assemble on two-dimensional materials makes them promising candidates for biosensing applications.^11,30−32

Methods and Experimental Section

Materials

Peptides (powder purity >95%) were purchased from COSMO BIO Co., Ltd., Japan. Peptide powder was dissolved in Milli-Q water to make a 1 mM stock solution. The stock solution was stored frozen at −20 °C and diluted to 1–50 μM as needed.

Hemin powder (Fuji Film) was dissolved in NaOH 25-mM solution (Wako Pure Chemicals Co.) to be 1 mM and subsequently diluted with Milli-Q water to the desired concentration. The phosphate buffer solution (PB) (100 mmol/L phosphate buffer, pH = 7.0) was purchased from Wako Pure Chemicals Co.

AFM Samples

For ex situ AFM measurements, graphite flakes cleaved with Scotch tape were mechanically transferred onto a silicon wafer with a 300 nm layer of silicon dioxide (SUMCO, Japan). 100-μL peptide solution (1 μM) was then placed on graphite for 1 h under a humid condition. The peptide solution was then gently blown off with nitrogen gas, and the samples were dried overnight in a vacuum desiccator to remove any remaining solution. For in situ AFM measurements, substrates of highly oriented pyrolytic graphite (HOPG) were used. The surface was mechanically cleaved with Scotch tape to make a fresh surface, and the 100 μL peptide solution was placed on it for 1 h before the AFM measurement. Hemin immobilization on self-assembling peptides was prepared by replacing the peptide solution with deionized water (DI water) followed by injection of the hemin solution.

Amplitude Modulation-Atomic Force Microscopy (AM-AFM)

Peptide self-assembled structures on graphite surfaces were measured by atomic force microscope (MFP-3D-SA, Asylum Research, Oxford Instruments) in air. The AFM instrument was equipped with a silicon cantilever (OMCL-AC160TS, Olympus, JP) with a resonance frequency of 300 ± 100 kHz and a spring constant of 26 N/m. Gwyddion (Czech Metrology Institute, CZ) was used for AFM image analysis.

Frequency Modulation-Atomic Force Microscopy (FM-AFM)

The high-resolution AFM images of peptide self-assemblies on the HOPG were obtained using a custom-built frequency modulation AFM (FM-AFM) system operating in liquid environments, and equipped with an ultralow-noise cantilever deflection sensor.¹³ The oscillation of the cantilever was driven by photothermal excitation at resonance with the amplitudes set at <0.25 nm. The AFM scanning process was controlled using a commercial SPM controller (ARC2, Asylum Research). A constant cantilever oscillation amplitude was maintained by adjusting the excitation signal amplitude using a commercially available controller (OC4, SPECS). The AFM was operated in the constant frequency shift (Δf) mode, where the tip–sample distance was adjusted such that Δf was kept constant. AFM image data were collected using a commercially available silicon cantilever (160AC-NG, OPUS) with a nominal spring constant of 26 N/m and a nominal apex radius of ∼7 nm. We obtained cantilever eigen frequencies in the range of 125–142 kHz, and quality factors of 7–8 in Milli-Q water. The cantilever spring constants were calibrated using the thermal noise method after each experiment. AFM data were processed and analyzed using the WSxM image analysis software. The raw AFM data were postprocessed by flattening and plane-fitting to eliminate background tilt where necessary. The scanning parameters, including the scan angle, imaging set point, and rate, were varied to obtain the optimum contrast of the surface features in the AFM images.

High-Speed Atomic Force Microscopy (HS-AFM)

Using a custom-built high-speed atomic force microscope (HS-AFM) developed by Ando et al.,¹⁴ we measured the dynamics of hemin on (YH)₄ peptide assemblies (Movie S1). The HS-AFM technique employs tapping mode with amplitude modulation feedback and operates in solution using ultrashort cantilevers (USC-F1.2-K0.15; NANOANDMORE). The cantilevers have a spring constant of approximately 0.15 N/m, a resonant frequency of ∼1.2 MHz, and a quality factor of ∼3.0 in air. For HS-AFM imaging, the cantilever peak-to-peak free oscillation amplitude (A₀) was set to approximately 1.6–1.8 nm, with the feedback amplitude set-point adjusted to ∼0.9 A₀. The AFM image rendering and data analysis were carried out with custom-made analysis software, UMEX Viewer, as previously described.^15,16

Preparation of Electrodes for Electrochemistry

A substrate of HOPG was mounted on a glass slide and connected to a conductive wire with silver paste. On the HOPG surface, 100 μL of 2-μM peptide solution was placed for 1 h and 100 μL of 50 μM hemin solution was then replaced with the peptide solution. The hemin solution was then rinsed with 100-mM PB after 1-h incubation. Following this, the HOPG-peptide-hemin electrode was dried by pipetting out the solution.

Electrochemical Measurements

Electrochemical measurements were performed in a three-electrode system using a Versastat 3 (Princeton Applied Research), with Ag/AgCl as the reference electrode and platinum wire as the counter electrode. The working electrode was the prepared HOPG-peptide-hemin electrode. The PB solution was held on the working electrode by a handmade PDMS well (Supporting Information, Figure S1). The solution of 100-mM phosphate buffer (pH 7) was purged with nitrogen gas for at least 20 min beforehand to remove oxygen dissolved in the solution. Furthermore, the nitrogen gas was continuously introduced to the solution during the measurement to prevent the dissolving of oxygen from the air. For the cyclic voltammetry measurements, the range of the electrode potential was −0.8 to 0.5 V.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c15373.

• Schematic diagram of the electrochemical measurement system; peptide unit cell calculated from AFM images; concentration dependence of peptide structure; disorder of peptide structures on graphite; immobilized hemin on self-assembling peptides; structure of immobilized hemin on self-assembling peptides; X-ray photoelectron spectroscopy (XPS) characterization; and stability of peptide self-assembles structures (PDF)

• High-speed AFM shown in Figure 3d (Movie S1) (MOV)

The authors declare no competing financial interest.