# TOWARDS UNVEILING THE HIDDEN DYNAMICS OF BACTERIORHODOPSIN WITH MEMS-BASED ATOMIC FORCE MICROSCOPY

Suyambulingam Subramanian, Nicolas Mauran, Guillaume Jourdan, Ignacio Casuso, Bernard Legrand

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Suyambulingam Subramanian, Nicolas Mauran, Guillaume Jourdan, Ignacio Casuso, Bernard Legrand. TOWARDS UNVEILING THE HIDDEN DYNAMICS OF BACTERIORHODOPSIN WITH MEMS-BASED ATOMIC FORCE MICROSCOPY. 2025 23rd International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers), Jun 2025, Orlando, United States. pp.817-820, 10.1109/Transducers61432.2025.11110349 . hal-05251930

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# TOWARDS UNVEILING THE HIDDEN DYNAMICS OF BACTERIORHODOPSIN WITH MEMS-BASED ATOMIC FORCE MICROSCOPY

Suyambulingam Subramanian¹, Nicolas Mauran¹, Guillaume Jourdan², Ignacio Casuso³,
Bernard Legrand¹

¹LAAS, CNRS, Université de Toulouse, FRANCE, ²CEA-LETI, Université Grenoble-Alpes,
FRANCE, ³DyNaMo, INSERM, Université Aix-Marseille, FRANCE

## ABSTRACT

Understanding membrane protein unfolding is essential for elucidating protein stability and function. Here, we employ MEMS-based atomic force microscopy (AFM) to investigate the unfolding dynamics of bacteriorhodopsin (BR) with microsecond resolution. Compared to conventional cantilevers, High frequency MEMS probes minimize meniscus effects and enable high-sensitivity force spectroscopy under dry conditions. Unfolding force-extension curves reveal discrete structural transitions consistent with BR domain unfolding. These findings demonstrate MEMS-AFM as a valuable tool for high-speed biomolecular force measurements. This study also lays the groundwork for implementing optomechanical probes at > 100 MHz, advancing time-resolved protein mechanics into the nanosecond regime.

## KEYWORDS

AFM, MEMS-AFM, Optomechanical probes, High speed AFM.

## INTRODUCTION

Atomic Force Microscopy (AFM) has become a vital tool for studying surface morphology and mechanical properties of biological molecules including proteins [1]. The ability of AFM to single molecule force spectroscopy has significantly aided our understanding of protein unfolding dynamics and membrane biophysics [2]. However, studying complex biological process such as conformational dynamics of protein unfolding requires AFM techniques with enhanced temporal resolution [3]. Traditional Cantilevers, face fundamental limitations in achieving the required resolution due to constraints imposed by optical detection and mechanical bandwidth.

One of the primary challenges in high-speed AFM is reaching high resonance frequencies while maintaining optimal force sensitivity. Conventional AFM detects cantilever motion via optical beam deflection or interferometry [4]. However, as cantilevers are shrunk to increase their resonance frequency, the optical diffraction limit is approached, resulting in decreased detection resolution. This fundamental restriction limits further cantilever downsizing and is a bottleneck for enhancing the time resolution of AFM devices. To address these limitations, alternate sensing methods, such as microelectromechanical systems (MEMS)-based probes with built-in transducers, have emerged as attractive possibilities for next-generation AFM technology.

MEMS-based AFM probes has various advantages over traditional cantilevers, including higher resonance

frequencies and seamless integration with sophisticated actuation and sensing systems. Unlike traditional cantilevers, MEMS-based resonators use in-plane vibration modes, capacitive actuation, which eliminates the need for optical detection [5]. These probes function at resonance frequencies greater than 10 MHz, overcoming the constraints of cantilever- and quartz tuning fork-based AFM devices [6].

In this study, we use MEMS-based AFM probes to analyze the dynamic unfolding behavior of bacteriorhodopsin (BR), a membrane protein crucial for light-driven proton transport. Many essential biomolecular processes occur on timescales that traditional cantilevers cannot reach due to resonance frequency restrictions. These brief molecular events, predicted by molecular dynamics simulations, are critical for completely understanding the operation and function of biomolecules. Our goal is to explore these ultrafast regimes using MEMS-based AFM and in future with optomechanical devices [7].

## PROTEIN UNFOLDING DYNAMICS

Understanding protein unfolding pathways is critical for understanding the fundamental principles of protein stability and function, as well as misfolding-related disorders like Alzheimer's and Parkinson's [8]. The unfolding process reveals important information on the energy landscape of proteins, the presence of intermediate states, and the forces required to destroy native structures [9]. Identifying these intermediate states is critical for understanding folding mechanisms, since they frequently represent functional or misfolded conformations with important biological ramifications.

<figure><img src="image_2.png" /><figcaption>Figure 1: Implication of Time Resolution in Protein Dynamics. This figure illustrates the significance of time resolution in capturing protein unfolding dynamics. Higher time resolution enables the detection of short-lived intermediate states, providing deeper insights into the energy landscape and transition kinetics of biomolecular processes</figcaption></figure>

Bacteriorhodopsin (BR) has emerged as an appropriate
model system for investigating membrane protein unfolding due to its well-defined structure and functional significance in proton transport [10]. Previous AFM research found numerous unfolding intermediates in BR, highlighting its complexity and importance in membrane protein biophysics. However, traditional AFM techniques have struggled to capture rapid unfolding transitions due to their limited temporal resolution [11]. Although recent advances, like ultrashort cantilevers, have increased the time resolution of AFM-based unfolding investigations to sub-milliscond levels by increasing resonance frequencies [12], hurdles remain in resolving microsecond-scale changes. Given that many crucial conformational changes in proteins occur on such short timeframes, improving the time resolution of AFM is critical for precisely mapping protein unfolding routes (Figure 1).

In this study, we use MEMS-based AFM to analyze the unfolding dynamics of BR at unparalleled time resolution. MEMS probes outperform traditional cantilevers by achieving greater resonance frequencies and displacement sensitivity, paving the way to microsecond-scale temporal resolution [5]. Compared to cantilevers and quartz tuning forks, MEMS technology offers a promising tradeoff between resolution, stability, and operational efficiency for a wide range of biological processes (Fig. 2).

<figure><img src="image_3.png" /><figcaption>Figure 2: Comparison of Cantilever, Quartz, MEMS, and Optomechanical Probes. This figure compares different AFM probe technologies based on key characteristics such as time resolution, mechanical sensitivity, operational frequency, and amplitude. The chart also highlights the biological processes that can be investigated at various timescales, emphasizing the advantages of MEMS-based probes for high-speed single-molecule studies.</figcaption></figure>

## MEMS PROBES

For this study, we utilized MEMS-based ring resonator probes obtained from VMicro. The design, fabrication, and operational principles of these probes have been extensively documented in prior studies [6].

MEMS ring resonators provide a transformative approach to high-speed AFM by integrating on-chip capacitive transduction for both actuation and readout. This eliminates the need for traditional optical detection, allowing for significantly enhanced displacement sensitivity, reaching the thermomechanical noise limit. The use of microwave reflectometry further refines the detection mechanism, enabling sub-nano Newton force resolution [6,13]. These probes typically operate at resonance frequencies of approximately 15 MHz, with

quality factors around 500, achieving a force resolution below pN/√Hz. Their exceptionally small vibration amplitude, in the range of 10 pm, ensures minimal perturbation of the sample during measurement [14,15].

One of the major challenges of MEMS probes is their operation in liquid environments. Immersion and presence of liquid in the capacitive airgap may disrupt capacitive actuation and readout, restricting their use and reliability. To circumvent this limitation, we focused our experiments on dry or ambient-air conditions. Bacteriorhodopsin (BR) was specifically chosen for this study due to its stability and functional activity even under dry conditions, making it an ideal candidate for force spectroscopy measurements without requiring immersion in liquid.

However, even in dry conditions, the presence of a native thin water layer on the BR membrane introduces meniscus interactions, which can significantly influence AFM measurements. In conventional cantilever-based AFM, the meniscus effect poses several challenges, but unique operation of MEMS probes overcome these challenges. As illustrated in Figure 3, MEMS probes oscillate continuously within the water meniscus due to their ultra-small amplitudes (fm–pm range), unlike conventional cantilevers, which repeatedly enter and exit the meniscus during each oscillation cycle. This uninterrupted operation prevents large perturbations in tip-sample interactions and enhances the detection of minute unfolding events in BR membranes.

<figure><img src="image_4.png" /><figcaption>Figure 3: Schematics of Dynamic Mode AFM in Dry Bacteriorhodopsin Samples with a Thin Native Water Layer. (a) Cantilever-Based AFM: High-amplitude (Å-nm) oscillations disrupt the meniscus, introducing strong adhesion effects that obscure molecular contributions from the protein and membrane. (b) MEMS Probe AFM: Low-amplitude (fm-pm) oscillations preserve the meniscus structure while maintaining exceptional displacement sensitivity, allowing precise detection of protein and membrane mechanics.</figcaption></figure>

## RESULTS & DISCUSSIONS

To establish a reference for protein-specific unfolding behavior, control experiments were conducted on freshly cleaved mica and silicon surfaces under identical humidity conditions (40% relative humidity). Atomic force microscopy (AFM) imaging of mica revealed an atomically flat surface with minimal roughness (Fig. 4a). The force-distance spectroscopy curve obtained on mica (Fig. 4b) exhibited a characteristic adhesion width of approximately 5 nm, indicative of weak tip-surface interactions. This observation is consistent with the hydrophilic nature of mica, where adhesion is primarily governed by capillary forces, as evidenced by the linear trend in the adhesion region. Whereas, AFM measurements on silicon surfaces
revealed significantly stronger tip adhesion, with an adhesion width of approximately 27 nm (Fig. 5b). This increase is attributed to enhanced capillary interactions due to silicon's native oxide layer, which is inherently more hydrophobic than mica. The significant change in adhesion widths between the two surfaces underscores the role of surface nature in AFM-based adhesion studies and establishes essential reference data for subsequent bacteriorhodopsin (BR) measurements.

<figure><img src="image_5.png" /></figure>

Figure 4: AFM Characterization of Mica Surface. (a) AFM topography of the mica surface at 40% humidity, showing an atomically flat morphology with minimal particulate features, allowing an estimate of the AFM tip radius (< 50 nm). (b) Representative force-distance curve of mica, showing an adhesion width of ~5 nm, indicative of weak tip-surface interactions. The linear response in the adhesion region confirms that the interaction is predominantly meniscus-driven.

<figure><img src="image_6.png" /></figure>

Figure 5: AFM Characterization of Silicon Surface. (a) AFM topography of silicon at 40% humidity, revealing a smooth morphology. (b) Force-distance curve showing a significantly larger adhesion width (~27 nm), indicative of stronger capillary forces and hydrophobic surface characteristics. Similar to mica, the adhesion response follows a linear trend, confirming meniscus-driven interactions.

Following the control experiments, BR membrane patches were deposited onto freshly cleaved mica and dried. AFM imaging using a MEMS AFM probe was performed to confirm membrane integrity. Large-area AFM imaging (Fig. 6a) revealed well-distributed BR patches across the substrate. Higher-resolution imaging (Fig. 6b) of individual BR patches displayed well-defined membrane structures, with thickness measurements (Fig. 6c) confirming a value of approximately 5 nm, consistent with previous reports of single-layer BR membranes. These imaging results validate the successful preparation of BR samples and provide a stable substrate for force spectroscopy measurements.

To investigate the unfolding dynamics of BR, force spectroscopy was performed using a controlled tip approach and retraction sequence. The AFM tip was brought into contact with the BR membrane at a velocity of 0.0006 nm/µs, maintaining contact for 10 ms to promote

adhesion of BR molecules. Retraction occurred in two phases: 1st Slow retraction (0.0003 nm/µs) to capture unfolding events. Then Rapid detachment (0.01 nm/µs) to ensure complete separation.

<figure><img src="image_7.png" /></figure>

Figure 6: AFM Imaging of BR Membranes at 40% humidity. (a) Large-area AFM topography showing BR membrane patches on mica. (b) High-resolution image of an individual BR patch, highlighting its well-defined morphology. (c) z-profile measurement confirming a membrane thickness of ~5 nm, indicative of a BR monolayer.

The amplitude versus distance curves obtained during these experiments (Fig. 7b) exhibited distinct unfolding-like events, characterized by saw tooth pattern indicative of sequential structural transitions within the BR protein. Notably, these unfolding signatures were absent in control experiments on mica and silicon, confirming that they arise from protein-specific interactions rather than nonspecific tip adhesion.

<figure><img src="image_8.png" /></figure>

Figure 7: AFM Force Spectroscopy on BR Membranes at 40% humidity. (a) Schematic representation of the approach and retract process. (b) Representative amplitude vs. distance curve obtained during AFM spectroscopic measurements on BR membrane patches, showing discrete unfolding events unique to BR.

To assess the influence of the BR membrane on tip-sample interactions, we compared the normalized retraction curves obtained from control surfaces (mica and silicon) with those from BR-coated samples, both with and without unfolding events. This comparison provides insights into the adhesion characteristics and mechanical response of BR under force spectroscopy conditions (Fig. 8). For control surfaces: Mica, Displays an adhesion width of ~5 nm, consistent with weak tip-sample interactions due to its hydrophilic nature. Silicon, exhibits an adhesion width of ~27 nm, indicating stronger capillary interactions arising from its hydrophobic native silicon. For BR-coated mica: The adhesion response deviates from the linear trend observed on control surfaces, following a third-order polynomial, suggesting a complex interplay between the AFM tip, BR membrane, and hydration forces. The adhesion width increases to ~12 nm, reflecting additional interactions introduced by the BR membrane. When unfolding events occur, the adhesion width further
increases to ~19 nm, suggesting molecular interactions beyond simple membrane adhesion, most likely due to the mechanical unfolding of BR protein domains. A characteristic sawtooth pattern is evident in force curves obtained from BR samples exhibiting unfolding events, a hallmark of sequential unfolding of protein domains under mechanical force. This pattern aligns with single-molecule force spectroscopy studies of membrane proteins, confirming that we are probing BR unfolding at the molecular level [10].

<figure><img src="image_9.png" /><figcaption>Figure 8: AFM Retract Curve Comparison. Normalized retract curves for mica (blue) and silicon (orange) show a linear response with adhesion widths of ~5 nm and ~27 nm, respectively. The BR-coated mica surface exhibits a third-order polynomial dependence (green) with an adhesion width of ~12 nm. When unfolding events are detected (red), the adhesion width increases to ~19 nm. The inset highlights the sawtooth pattern, characteristic of sequential BR unfolding.</figcaption></figure>

The distribution of corelated force peaks observed in BR unfolding curves suggests molecular unfolding events. Key unfolding distances include: at ~30 nm, corresponding to the ED pair unfolding. And at ~42 nm attributed to the CB pair unfolding. Finally, at ~60 nm, associated with the unfolding of helix A in BR. These distances are in close agreement with previously reported single-molecule force spectroscopy data on BR [12], further validating that we are directly probing molecular-level unfolding events using MEMS probes.

## CONCLUSION

These results underscore the effectiveness of MEMS-based AFM probes in detecting protein unfolding events with high time resolution. Compared to conventional cantilevers, MEMS probes operate at higher frequencies and lower amplitudes, significantly reducing perturbation from meniscus forces while enabling high time resolved force spectroscopy in ambient air condition. Furthermore, this study establishes a foundation for the future implementation of optomechanical probes operating at ultra-high frequencies above 100 MHz in both dry and liquid environments [7]. Such advancements would enable nanosecond-scale time resolution, opening new avenues for exploring the ultrafast dynamics of membrane protein mechanics at previously inaccessible temporal scales. The insights gained from this work not only enhance our understanding of BR unfolding but also pave the way for next-generation force spectroscopy techniques with unprecedented speed and precision.

## ACKNOWLEDGEMENTS

This work was supported by the French National Research Agency (ANR) under the research project HERMES (grant ANR-21-CE42-0031) and by the RENATECH French national technological network.

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## CONTACT

*Suyambulingam Subramanian, ssubramani@laas.fr*