At Hayamizu Lab, we conduct research at the interface that connects the world of biology with electronics. Biological systems sustain life through dynamic activity of biomolecules. Biosensors are devices that convert the movement of these biomolecules into electrical signals. To detect diverse biological activities, it is essential to understand both the structure of biomolecules and the electronic state at the solid surface simultaneously. We tackle this challenge by utilizing graphene, a representative nanomaterial, as the electronic material.

In biosensor development, integrating biomolecules with electronic devices is crucial. Two-dimensional materials, such as graphene, are well-suited for studying biomolecular structures and the electronic states on solid surfaces due to their atomically flat surfaces and excellent electrical properties.

It is known that designed self-assembling peptides form well-ordered monolayer structures on two-dimensional materials. The self-assembled structures, when built on layered materials, alter the electrical properties of graphene. Through surface modification with peptides, these materials hold promise for use in biosensing applications.

Understanding the electronic states at the interface created by peptide self-assembly on two-dimensional materials is a key step toward realizing controlled nano-bioelectronics.

Hayamizu Y., et al., Scientific Reports, 2016, 6(1), 33778.
Li P., et al., ACS Applied Materials & Interfaces, 2019, 11(23), 20670-20677.

Observing peptide self-assembly and understanding its mechanisms are fundamental for designing peptides and developing sensors.

Amino acids and peptides have chirality, and stereochemical matching plays a crucial role in the two-dimensional self-assembly process. We studied the chiral recognition of L- and D-peptides on MoS₂ and proposed a lattice-matching model. This allows for the rational design of peptide self-assembled structures with desired functions.

Approaches such as peptide sequence design, electrochemical stimulation, pH effects, and fluid control can deepen our understanding of peptide surface self-assembly at the solid-liquid interface. For instance, we investigated the effects of organic solvents on the self-assembly of peptides designed to mimic silk fibroin. It was shown that solvent mixtures suppress peptide self-assembly, with a correlation between solvent dielectric constants and threshold concentrations.

Sun L., et al., Langmuir, 2021, 37(29), 8696-8704.
Ccorahua R., et al., The Journal of Physical Chemistry B, 2021, 125(39), 10893-10899.

Self-assembling peptides are known to form well-ordered structures on 2D materials like mica, graphene, and MoS₂. These peptides hold potential as molecular scaffolds for biosensing based on two-dimensional materials. However, the stability of these self-assembled structures in aqueous environments remains to be evaluated.

We developed peptides that mimic silk protein sequences and can maintain ordered nanostructures even after being rinsed with water. These peptides remained stable under applied voltage, making them valuable as scaffold molecules for practical biosensing.

Our peptide acquired the ability to immobilize probe molecules for biosensing and prevent non-specific adsorption via co-assembly. Interestingly, the self-assembled structures exhibited two structural phases, with only one phase showing affinity for target molecules.

Sun L., et al., RSC Advances, 2016, 6(99), 96889-96897.
Li P., et al., ACS Applied Materials & Interfaces, 2019, 11(23), 20670-20677.

Research on protein crystals has played a significant role in understanding their functions by elucidating their structures. On the other hand, with peptides, which are shorter and simplified versions of proteins, we can directly link function and structure through simplification, aiming for applications such as electronics.

We controlled crystallization on substrates using dipeptides containing phenylalanine and asparatic acid. It was revealed that solution shearing, a method of drying peptide solution on a substrate, resulted creating a peptide thin film on the substrate with a higher crystal alignment and more limited crystal forms compared to other methods.

We also conducted research using peptides inspired by diphenylalanine, which has been studied in the context of amyloid-β. By incorporating glutamic acid alongside the phenylalanine with aromatic rings, we demonstrated the formation of a dense hydrogen-bonding network between molecules. This was revealed through Raman spectroscopy, X-ray diffraction (XRD), and density functional theory (DFT) calculations. The relationship between Raman spectroscopy and XRD results showed a trend different from previously known relationships, suggesting new possibilities for peptide crystals.

Motai K., et al., Crystal Growth & Design, 2023, 23(6), 4556-4561.
Motai K., et al., Journal of Materials Chemistry C, 2020, 8(25), 8585-8591.

A biosensor is a device that detects specific chemical substances or biomolecules with high speed and sensitivity. By combining biological materials (such as enzymes, antibodies, nucleic acids) with a physical sensor, biosensors can convert biological reactions into chemical or physical signals, enabling the quantitative measurement of target substances. The molecular recognition capabilities of the biological materials allow biosensors to detect their targets with high selectivity and sensitivity. A biosensor consists of two main components: a biological recognition element (bioreceptor) and a signal transduction element (transducer).

Biosensors are widely used in our daily lives. For instance, glucose sensors for measuring blood sugar levels and antibody test kits for detecting viral infections are both types of biosensors. Biosensors play important roles not only in clinical diagnostics and drug discovery but also in various fields such as healthcare, environmental monitoring, and food quality control.

In biosensors using two-dimensional nanomaterials, as shown in the figure, the interface formed by the two-dimensional nanomaterials and immobilized bioreceptor functions as the transducer. The bioreceptor selectively captures the target substance in the sample, and the resulting biological or chemical changes are converted into electrical signals for output. Although it is already known that two-dimensional nanomaterials serve as excellent sensing platforms, the design of this interface is currently the most critical technical challenge in achieving practical, high target selectivity and sensitivity.

We have demonstrated biosensors functionalized with peptides on the surfaces of graphene and monolayer MoS₂. We successfully detected target substances selectively in solution, revealing that peptide molecular scaffolds hold great potential as transducers.

Tezuka S., et al., 2D Materials, 2020, 7(2), 024002.
Noguchi H., ACS Applied Materials & Interfaces, 2023, 15(11), 14058-14066.
Khatayevich D., et al., Small, 2014, 10(8), 1505-1513.

The human sense of smell is made possible by approximately 400 types of proteins called olfactory receptors, which are present in the cells of the nose. The reason why products that replicate human olfaction are not commonly sold in electronics stores is that achieving such replication has been extremely difficult.

An odor sensor is a device that selectively and sensitively detects odor molecules, which are volatile organic compounds (VOCs). It is said that humans can perceive around 400,000 different types of odor molecules. Until now, gas chromatography-mass spectrometry (GC-MS) has been the primary method used for detecting VOCs. While this is a highly reliable detection method, it has limitations in sensitivity, and the relatively large size of the equipment makes it difficult to use for general purposes. Therefore, we have focused on developing a small, highly sensitive odor sensor by functionalizing the surface of a graphene field-effect transistor (GFET) with peptides.

This graphene-based odor sensor is fabricated by self-organizing peptides with receptors onto the surface of the GFET's graphene. The peptide sequences contain two functions: (1) a molecular scaffold for self-organization on the graphene surface, and (2) a bioreceptor that binds to odor molecules.

Using this approach, we successfully detected odor molecules, such as limonene and its optical isomers, with high selectivity and sensitivity. In the future, we expect that by increasing the number of GFET arrays and using a variety of peptide bioprobes, combined with machine learning techniques, it will be possible to classify odor molecules similarly to humans.

Rungreungthanapol T., *Analytical Chemistry*, 2023, 95(9), 4556-4563.

Yamazaki Y., et al., *ACS Applied Materials & Interfaces*, 2024, 16(15), 18564-18573.

Homma C., et al., *Biosensors and Bioelectronics*, 2023, 224, 115047.

Research on enzyme sensors plays an important role in the fields of biosensors and electrochemistry. In particular, materials that function similarly to enzymes are being developed, and self-assembling peptide-based sensors are gaining attention. Peptides are used as materials to detect specific molecules or ions and are notable for their ease of design and synthesis. For example, short peptides can be self-assembled on graphite surfaces to achieve highly efficient catalytic reactions comparable to those of natural enzymes. The development of peptide-based artificial enzyme sensors offers a new technology that improves the efficiency of chemical and energy conversions, and these sensors are expected to be widely used in the future as environmentally friendly sensor materials.

Luo W., et al., Nanoscale, 2022, 14(23), 8326-8331.

Luo W., et al., Langmuir, 2023, 39(20), 7057-7062.

At Hayamizu Laboratory, we are also conducting research to develop technologies for measuring and observing bio-nano interfaces. This includes fundamental research on electronics using two-dimensional materials, such as graphene and transition metal dichalcogenides (TMDs), which are widely studied. Through this research, we aim to contribute to advancements in various fields of bioelectronics, including biosensors.

Molybdenum disulfide (MoS₂), which has a structure similar to graphene, has attracted attention as a two-dimensional semiconductor material with unique electrical and optical properties. Controlling the number of layers of MoS₂ is essential for device applications. To address this, we developed a laser-induced electrochemical etching method, combining laser etching and electrochemical etching techniques, whose effectiveness has been demonstrated with other materials in prior research. This method enabled the precise production of monolayer MoS₂ from multilayer MoS₂.

Additionally, we investigated the modulation of electron density in graphene transistors using organic semiconductor molecules with various HOMO levels, aiming to control the electron concentration in graphene via adsorbed organic molecules. Our findings revealed a strong correlation between the HOMO level of the organic molecules and the doping effect.

These insights are useful for examining how nanomaterials are electrically influenced by molecules adsorbed on the surfaces of all nanomaterials.

Sunamura K., et al., Journal of Materials Chemistry C, 2016, 4(15), 3268-3273.

Masujima H., Journal of Electronic Materials, 2017, 46, 4463-4467.

The phenomenon of liquid-liquid phase separation (LLPS) refers to the process in which substances like water and oil separate within a liquid to form droplets. It has been increasingly recognized in the field of life sciences that this liquid-liquid phase separation plays a critical role within cells.

In the field of molecular biology, it has been revealed that poly(PR) dipeptide repeats, which are produced by abnormalities in the C9orf72 gene, contribute to the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through liquid-liquid phase separation. Particularly, the influence of poly(PR) on intracellular biochemical reactions through LLPS has garnered attention. We investigated this mechanism by focusing on the interactions between molecules.

As part of our experimental approach, we generated mutants with different sequences of poly(PR) and observed their LLPS behavior. Specifically, we varied the number of P and R residues to create peptides such as PRPR and PPRR, altering the length of each amino acid sequence. In addition, we conducted quantitative proteome analysis to identify proteins interacting with poly(PR). Furthermore, using fluorescence recovery after photobleaching (FRAP) with fluorescence microscopy, we evaluated the diffusion properties of poly(PR) within cells.

As a result, poly(PR) with adjacent arginine and proline residues exhibited the highest cytotoxicity. Poly(PR) interferes with intracellular biochemical reactions through multivalent interactions with positively charged proteins via LLPS. Notably, it was confirmed that the phase-separated droplets formed by poly(PR) hindered the transcription, translation, and diffusion of the nuclear protein NPM1. These findings provided new insights into the role of poly(PR) in the pathology of ALS.

This research has contributed to establishing a foundation for the development of treatments for ALS and FTD by elucidating the properties of poly(PR) and its effects on neuronal cells.

Chen C., et al., Langmuir, 2021, 37(18), 5635-5641.

Chen C., et al., Journal of Cell Biology, 2021, 220(11), e202103160.