Peptide Monolayers Research Articles

Detection of a Chirality-Induced Spin Selective Quantum Capacitance in α-Helical Peptides.

Advanced Kelvin probe force microscopy simultaneously detects the quantum capacitance and surface potential of an α-helical peptide monolayer. These indicators shift when either the magnetic polarization or the enantiomer is toggled. A model based on a triangular quantum well in thermal and chemical equilibrium and electron-electron interactions allows for calculating the electrical potential profile from the measured data. The combination of the model and the measurements shows that no global charge transport is required to produce effects attributed to the chirality-induced spin selectivity effect. These experimental findings support the theoretical model of Fransson et al. Nano Letters 2021, 21 (7), 3026-3032. Measurements of the quantum capacitance represent a new way to test and refine theoretical models used to explain strong spin polarization due to chirality-induced spin selectivity.

Surface-acoustic-wave-driven graphene plasmonic sensor for fingerprinting ultrathin biolayers down to the monolayer limit

Surface plasmon polaritons in graphene can enhance the performance of mid-infrared spectroscopy, which is key for the study of both the composition and the conformation of organic molecules via their vibrational resonances. In this paper, a plasmonic biosensor using a graphene-based van der Waals heterostructure on a piezoelectric substrate is theoretically demonstrated, where far-field light is coupled to surface plasmon-phonon polaritons (SPPPs) through a surface acoustic wave (SAW). The SAW creates an electrically-controlled virtual diffraction grating, suppressing the need for patterning the 2D materials, that limits the polariton lifetime, and enabling differential measurement schemes, which increase the signal-to-noise ratio and allow a quick commutation between reference and sample signals. A transfer matrix method has been used for simulating the SPPPs propagating in the system, which are electrically tuned to interact with the vibrational resonances of the analytes. Furthermore, the analysis of the sensor response with a coupled oscillators model has proven its capability of fingerprinting ultrathin biolayers, even when the interaction is too weak to induce a Fano interference pattern, with a sensitivity down to the monolayer limit, as tested with a protein bilayer or a peptide monolayer. The proposed device paves the way for the development of advanced SAW-assisted lab-on-chip systems combining the existing SAW-mediated physical sensing and microfluidic functionalities with the chemical fingerprinting capability of this novel SAW-driven plasmonic approach.

Effect of Newly Synthesized Structures of Peptides on the Stability of the Monolayers Formed.

The aim of the study was to evaluate the effect of the peptide structure (WKWK)2-KWKWK-NH2, P4 (C12)2-KKKK-NH2, P5 (KWK)2-KWWW-NH2, P6 (KK)2-KWWW-NH2 on their physicochemical properties. The thermogravimetric method (TG/DTG) was used, which made it possible to observe the course of chemical reactions and phase transformations occurring during the heating of solid samples. Based on the DSC curves, the enthalpy of the processes occurring in the peptides was determined. The influence of the chemical structure of this group of compounds on their film-forming properties was determined using the Langmuir-Wilhelmy trough method and was followed by molecular dynamics simulation. Evaluated peptides showed high thermal stability and the first significant mass loss occurred only at about 230 °C and 350 °C. The analysis of the compressibility coefficient of individual peptides indicates that all formed peptide monolayers were in the expanded liquid phase. Their maximum compressibility factor was less than 50.0 mN/m. Its highest value of 42.7 mN/m was achieved in a monolayer made of P4. The results obtained in molecular dynamic simulation indicate that non-polar side chains played an important role in the properties of the P4 monolayer, and the same applies to P5, except that a spherical effect was observed here. A slightly different behavior was observed for the P6 and P2 peptide systems, where the type of amino acids present had an influence. The obtained results indicate that the structure of the peptide affected its physicochemical and layer-forming properties.

Impedimetric Bacterial Detection Using Random Antimicrobial Peptide Mixtures.

The biosensing of bacterial pathogens is of a high priority. Electrochemical biosensors are an important future tool for rapid bacteria detection. A monolayer of bacterial-binding peptides can serve as a recognition layer in such detection devices. Here, we explore the potential of random peptide mixtures (RPMs) composed of phenylalanine and lysine in random sequences and of controlled length, to form a monolayer that can be utilized for sensing. RPMs were found to assemble in a thin and diluted layer that attracts various bacteria. Faradaic electrochemical impedance spectroscopy was used with modified gold electrodes to measure the charge-transfer resistance (RCT) caused due to the binding of bacteria to RPMs. Pseudomonas aeruginosa was found to cause the most prominent increase in RCT compared to other model bacteria. We show that the combination of highly accessible antimicrobial RPMs and electrochemical analysis can be used to generate a new promising line of bacterial biosensors.

Self-assembly of cyclic peptide monolayers by hydrophobic supramolecular hinges

A novel supramolecular tryptophan hinge motif enables the self-assembly of nanotube monolayers by hierarchical non-covalent polymerisation of cyclic peptides in lateral contact.

Front Cover: Kinase Sensing Based on Protein Interactions at the Catalytic Site (Chem. Eur. J. 17/2022)

An electrochemical biosensor has been developed that is based on the interaction between the catalytic site of the kinase and the phosphorylation site of its substrate for detection. We adsorbed the phosphorylation site peptide of HDGF, a substrate of ERK2, on gold electrodes and were able to electrochemically sense ERK2 without ATP. Surface chemistry analysis showed that ERK2 was bound to the peptide monolayer, which changed the packing of the layer, resulting in increased permeability of redox-active species through the layer and electrochemical sensing. More information can be found in the Research Article by A. Friedler, S. Yitzchaik et al. (DOI: 10.1002/chem.202104227).

Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in a lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. We performed a detailed analysis of the interaction between the ELK-1 peptide and the kinase on gold surfaces. Atomic force microscopy, variable angle spectroscopic ellipsometry, X-ray Photoelectron Spectroscopy, and polarization modulation IR reflection-absorption spectroscopy analysis of the gold surface revealed the adsorbed layer of the ERK2 on the peptide monolayer. The sensors showed a high level of target selectivity for ERK2 compared to the p38γ kinase and BSA. ERK2 was detected in its cellular concentration range, 0.5–2.0 μM, and the limit of detection was calculated to be 0.35 μM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

Kinase Sensing Based on Protein Interactions at the Catalytic Site.

The role kinases play in regulating cellular processes makes them potential biomarkers for detecting the onset and prognosis of various diseases, including many types of cancer. Current kinase biosensors, including electrochemical and radiometric methods, rely on sensing the ATP-dependant enzymatic phosphorylation reaction. Here we introduce a new type of interaction-based electrochemical kinase biosensor that does not require any chemical labelling or modification. The basis for sensing is the interactions between the catalytic site of the kinase and the phosphorylation site of its substrate rather than the phosphorylation reaction. We demonstrated this concept with the ERK2 kinase and its substrate protein HDGF, which is involved in lung cancer. A peptide monolayer derived from the HDGF phosphorylation site was adsorbed onto a gold electrode and was used to sense ERK2 without ATP. The sensitivity of the assay was down to 10 nM of ERK2, corresponding with the range of its cellular concentrations. Surface chemistry analysis confirmed that ERK2 was bound to the HDGF peptide monolayer. This increased the permeability of redox-active species through the monolayer and resulted in ERK2 electrochemical sensing. Since our detection approach is based on protein-protein interactions and not on the enzymatic reaction, it can be further utilized for more selective detection of different types of enzymes.

Conformation- and phosphorylation-dependent electron tunnelling across self-assembled monolayers of tau peptides

We report on charge transport across self-assembled monolayers (SAMs) of short tau peptides by probing the electron tunneling rates and quantum mechanical simulation. We measured the electron tunneling rates across SAMs of carboxyl-terminated linker molecules (C6H12O2S) and short cis-tau (CT) and trans-tau (TT) peptides, supported on template-stripped gold (AuTS) bottom electrode, with Eutectic Gallium-Indium (EGaIn)(EGaIn) top electrode. Measurements of the current density across thousands of AuTS/linker/tau//Ga2O3/EGaIn single-molecule junctions show that the tunneling current across CT peptide is one order of magnitude lower than that of TT peptide. Quantum mechanical simulation demonstrated a wider energy bandgap of the CT peptide, as compared to the TT peptide, which causes a reduction in its electron tunneling current. Our findings also revealed the critical role of phosphorylation in altering the charge transport characteristics of short peptides; more specifically, we found that the presence of phosphate groups can reduce the energy band gap in tau peptides and alter their electrical properties. Our results suggest that conformational and phosphorylation of short peptides (e.g., tau) can significantly change their charge transport characteristics and energy levels.

Evidence for new enantiospecific interaction force in chiral biomolecules

Evidence for new enantiospecific interaction force in chiral biomolecules

Construction and Characterizations of Antibacterial Surfaces Based on Self-Assembled Monolayer of Antimicrobial Peptides (Pac-525) Derivatives on Gold

Background: Infection that is related to implanted biomaterials is a serious issue in the clinic. Antimicrobial peptides (AMPs) have been considered as an ideal alternative to traditional antibiotic drugs, for the treatment of infections, while some problems, such as aggregation and protein hydrolysis, are still the dominant concerns that compromise their antimicrobial efficiency in vivo. Methods: In this study, antimicrobial peptides underwent self-assembly on gold substrates, forming good antibacterial surfaces, with stable antibacterial behavior. The antimicrobial ability of AMPs grafted on the surfaces, with or without glycine spaces or a primer layer, was evaluated. Results: Specifically, three Pac-525 derivatives, namely, Ac-CGn-KWRRWVRWI-NH2 (n = 0, 2, or 6) were covalently grafted onto gold substrates via the self-assembling process for inhibiting the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Furthermore, the alkanethiols HS(CH)10SH were firstly self-assembled into monolayers, as a primer layer (SAM-SH) for the secondary self-assembly of Pac-525 derivatives, to effectively enhance the bactericidal performance of the grafted AMPs. The -(CH)10-S-S-G6Pac derivative was highly effective against S. aureus and E. coli, and reduced the viable amount of E. coli and S. aureus to 0.4% and 33.2%, respectively, after 24 h of contact. In addition, the immobilized AMPs showed good biocompatibility, promoting bone marrow stem cell proliferation. Conclusion: the self-assembled monolayers of the Pac-525 derivatives have great potential as a novel therapeutic method for the treatment of implanted biomaterial infections.

Effect of Hydrophobicity and Charge Separation on the Antifouling Properties of Surface-Tethered Zwitterionic Peptides.

Zwitterionic peptides emerge as a class of highly effective antifouling material in a wide range of applications such as biosensors, biomedical devices, and implants. We incorporated neutral amino acid spacers with different hydrophobicities, including serine (Ser), glycine (Gly), and leucine (Leu), into zwitterionic peptides with lysine-glutamic acid repeating units and investigated the structure and antifouling performance of the zwitterionic peptide brushes by surface plasma resonance, surface force apparatus (SFA), and all-atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that the hydrophilicity of neutral spacers alters the structure and antifouling performance of the peptide-modified surface. Hydrophilic Ser-inserted peptides reduced the interaction between the peptide monolayer and protein foulants, while hydrophobic Leu significantly increased the protein adhesion. SFA force measurements show that the presence of more spacers would increase the adhesion between the peptide monolayer and the modeling foulant lysozyme, especially for the hydrophobic spacers. MD simulations reveal that hydrophilic Ser spacers retain the hydrophilicity of the peptide monolayer and improve the antifouling performance, and Gly spacers give rise to more interchain cross-links. Leu spacers result in a more hydrophobic peptide monolayer, which leads to dehydration of the peptide monolayer and reduces the antifouling performances.

Hyaluronan (HA) Immobilized on Surfaces via Self-Assembled Monolayers of HA-Binding Peptide Modulates Endothelial Cell Spreading and Migration through Focal Adhesion.

The extracellular matrix (ECM) modulates a multitude of cell functions, and this regulation is provided by key ECM components forming a complex network. Hyaluronic acid (HA) is an abundant component of the ECM that binds to proteins and influences various activities of endothelial cells (ECs). Although the effect of soluble HA on cell spreading has been studied, the impact of peptide-bound HA has not yet been investigated in great detail. We aim to comprehensively study the roles of immobilized HA on the regulation of EC behavior compared to the more conventional use of soluble HA. A 2D model surface formed by self-assembled monolayers (SAMs) of a HA-binding peptide (Pep-1) is used as an anchor for HA immobilization. Mixed SAMs, consisting of thiolated Pep-1 and 1-octanethiol, are prepared and characterized by using ellipsometry and contact angle measurement. Full density Pep-1 SAMs are more hydrophilic and bind more HA than mixed SAMs. Cell spreading and migration are enhanced by immobilized low molecular weight (LMW) HA, which also facilitates cell alignment and elongation under laminar flow conditions and potentially drives directional migration. This effect is not mediated by the expression of CD44, and immobilized LMW HA is found to accelerate the assembly of focal adhesions. Such biomimetic surfaces provide new insights into the role of HA in regulating the spreading and phenotype of endothelial cells.

Simulating Peptide Monolayer Formation: GnRH-I on Silica.

Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomic level, which can be useful when attempting to interpret experiments or design new systems. The decapeptide gonadotrophin-releasing hormone I (GnRH-I) is known to control fertility in mammals for both sexes. It was previously shown that inoculation with silica nanoparticles (SiNPs) coated with GnRH-I makes an effective anti-fertility vaccine due to how the peptide adsorbs to the nanoparticle and is presented to the immune system. In this paper, we develop and employ a protocol to simulate the development of a GnRH-I peptide adlayer by allowing peptides to diffuse and adsorb in a staged series of trajectories. The peptides start the simulation in an immobile state in solution above the model silica surface, and are then released sequentially. This facile approach allows the adlayer to develop in a natural manner and appears to be quite versatile. We find that the GnRH-I adlayer tends to be sparse, with electrostatics dominating the interactions. The peptides are collapsed to the surface and are seemingly free to interact with additional solutes, supporting the interpretations of the GNRH-I/SiNP vaccine system.

Assemblies of bi-functional peptides on pyrolytic graphite for cell adhesion

Designing cell-electrode biointerfaces is important for electrochemical monitoring of cell functions and activities in their microenvironments. Herein, peptide monolayer was constructed on the pyrolytic graphite surface by self-assembling of tritryptophane, and the bi-functionalized peptide conjugating cell adhesion peptide, GRGDS (Gly-Arg-Gly-Asp-Ser) induced adhesion of anchorage-dependent cells. The peptide modification did not alter the electrochemical properties of pyrolytic graphite, and the redox signals of potassium ferricyanide were maintained. These observations indicate that non-covalent functionalization of graphite/graphene using bi-functional peptides would be effective for electrochemical measurement of cell activities.

Self-Assembled Pyrene Stacks and Peptide Monolayers Tune the Electronic Properties of Functionalized Electrolyte-Gated Graphene Field-Effect Transistors.

Aromatic molecules such as pyrenes are a unique class of building units for graphene functionalization, forming highly ordered π-π stacks while peptides provide more complex, biocompatible linkers. Understanding the adsorption and stacking behavior of these molecules and their influence on material properties is an essential step in enabling highly repeatable 2D material-based applications, such as biosensors, gas sensors, and solar cells. In this work, we characterize pyrene and peptide self-assembly on graphene substrates using fluorescence microscopy, atomic force microscopy and electrolyte-gated field-effect measurements supported by quantum mechanical calculations. We find distinct binding and assembly modes for pyrenes versus peptides with corresponding distinct electronic signatures in their characteristic charge neutrality point and field-effect slope responses. Our data demonstrates that pyrene- and peptide-based self-assembly platforms can be highly beneficial for precisely customizing graphene electronic properties for desired device technologies such as transport-based biosensing graphene field-effect transistors.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Reinforced Room-Temperature Spin Filtering in Chiral Paramagnetic Metallopeptides.

Chirality-induced spin selectivity (CISS), whereby helical molecules polarize the spin of electrical current, is an intriguing effect with potential applications in nanospintronics. In this nascent field, the study of the CISS effect using paramagnetic chiral molecules, which could introduce another degree of freedom in controlling the spin transport, remains so far unexplored. To address this challenge, herein we propose the use of self-assembled monolayers (SAMs) of helical lanthanide-binding peptides. To elucidate the effect of the paramagnetic nuclei, monolayers of the peptide coordinating paramagnetic or diamagnetic ions are prepared. By means of spin-dependent electrochemistry, the CISS effect is demonstrated by cyclic voltammetry and electrochemical impedance measurements for both samples. Additionally, an implementation of the standard liquid-metal drop electron transport setup has been carried out, and this process helped to demonstrate the peptides' suitability for solid-state devices. Remarkably, the inclusion of a paramagnetic center in the peptide increases the spin polarization as was independently proved by different techniques. These findings permit the inclusion of magnetic biomolecules in the CISS field and pave the way to their implementation in a new generation of (bio)spintronic nanodevices.

Grazing Incidence X-ray Diffraction Studies of Lipid-Peptide Mixed Monolayers during Shear Flow.

Grazing incidence X-ray diffraction (GIXD) studies of monolayers of biomolecules at an air–water interface give quantitative information of in-plane packing, coherence length of crystalline domains, etc. Rheo-GIXD measurements can reveal quantitative changes in the nanocrystalline domains of a monolayer under shear. Here, we report GIXD studies of monolayers of alamethicin peptide, DPPC lipid, and their mixtures at an air–water interface under steady shear stress. The alamethicin monolayer and the mixed monolayer show a flow jamming transition. On the other hand, the pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer under constant stress flows steadily with a notable enhancement of the area/molecule and coherence lengths, suggesting the fusion of nanocrystallites during flow. The DPPC–alamethicin mixed monolayer shows no significant change in the area/DPPC molecule, but the coherence lengths of the individual phases (DPPC and alamethicin) increase, suggesting that the crystallites of individual phases grow bigger by merging of domains. More phase separation occurs in the system during flow. Our results show that rheo-GIXD has the potential to explore in situ molecular structural changes under rheological conditions for a diverse range of confined biomolecules at interfaces.

Structures and Antifouling Properties of Self-Assembled Zwitterionic Peptide Monolayers: Effects of Peptide Charge Distributions and Divalent Cations

Zwitterionic peptides are great candidates as antifouling coating materials in many biomedical applications. We investigated the structure and antifouling properties of surface-tethered zwitterionic peptide monolayers with different peptide chain lengths and charge distributions using a combination of surface plasma resonance, atomic force microscopy, and all atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that zwitterionic peptides with more zwitterionic lysine (K) and glutamic acid (E) repeating units exhibit better antifouling performance. The block charge distributions of the positive and negative charges in the peptides (having multiple positive charges next to the same amount of negative charges), although affecting the structure of the peptide molecules, do not significantly change the antifouling properties of the peptide monolayers in the solutions containing monovalent ions. However, divalent cations, Ca2+ and Mg2+, in solution can significantly alter the structure and lower the antifouling performance of the zwitterionic peptide monolayers, especially with the sequences of block charges. All atomistic MD simulations quantitatively reveal that the divalent cations in solution lead to more interchain electrostatic cross-links between peptide chains, especially for peptides with block charges, which causes dehydration of the zwitterionic peptides and diminishes their antifouling performances.