Surface-Controlled Sialoside-Based Biosensing of Viral and Bacterial Neuraminidases.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein-electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

Using a Single Peptide to Electrochemically Sense Multiple Kinases.

Kinases are responsible for regulating cellular and physiological processes, and abnormal kinase activity is associated with various diseases. Therefore, kinases are being used as biomarkers for disease and developing methods for their sensing is highly important. Usually more than one kinase is involved in phosphorylating a target protein. However, kinase detection methods usually detect the activity of only one specific kinase. Here we describe an electrochemical kinase sensing tool for the selective detection of two kinases using the same target peptide. We demonstrate the sensing of kinases ERK2 and PKCδ. This is based on a single sensing element, a peptide that contains two distinct phosphorylation sites of these two kinases. Reversibility experiments with alkaline phosphatase and reaction with the electrochemically active ferrocene-labeled ATP showed that the mechanism of sensing is by detecting the enzymatic phosphorylation. Our approach can be further utilized to develop devices for the detection of multiple kinases and can be expanded to other types of enzymes involved in disease.

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.

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.

Kinase Sensing Based on Protein Interactions at the Catalytic Site.

Invited for the cover of this issue is Assaf Friedler, Shlomo Yitzchaik and co-workers at the Hebrew University of Jerusalem and the Academia Sinica. The image depicts a new approach for electrochemical kinase sensing that does not rely on phosphorylation. The kinase binds a peptide layer, which undergoes rearrangement, resulting in the permeation of redox-active species through the layer and electrochemical sensing. Read the full text of the article at 10.1002/chem.202104227.

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.

Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques.

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern.

Effect of Interfacial Properties on Impedimetric Biosensing of the Sialylation Process with a Biantennary N-Glycan-Based Monolayer.

Sensing enzymatic sialylation provides new tools for the evaluation of pathological events and pathogen invasion. Enzymatic sialylation is usually monitored via fluorescence or metabolic labeling, which requires relatively large amounts of the glycan substrate with limited availability. Using a label-free biosensor requires smaller quantities of substrates because the interactions induce measurable changes to an interface, which can be translated into a signal. The downside of label-free biosensors is that they are very sensitive to changes at the interface, and the properties of the surface layer can play a major role. Electrochemical impedance spectroscopy was used here to follow the enzymatic sialylation of a biantennary N-glycan acceptor in mixed monolayers. The surfaces contained either neutral, positively or negatively charged, or zwitterionic functional groups. The systems were characterized by contact potential difference, ellipsometry, and contact angle analyses. We found that the characteristics of the mixed monolayer have a profound effect on the biosensing of the enzymatic sialylation. Positively charged layers were found to adsorb the enzyme under the reaction conditions. Negatively charged and zwitterionic surfaces were nonresponsive to enzymatic sialylation. Only the neutral mixed monolayers provided signals that were related directly to enzymatic sialylation. This work demonstrates the importance of appropriate interface properties for monitoring enzymatic sialylation processes.

Determining the structure and binding mechanism of oxytocin-Cu2+ complex using paramagnetic relaxation enhancement NMR analysis.

Oxytocin is a neuropeptide that binds copper ions in nature. The structure of oxytocin in interaction with Cu2+ was determined here by NMR, showing which atoms of the peptide are involved in binding. Paramagnetic relaxation enhancement NMR analyses indicated a binding mechanism where the amino terminus was required for binding and subsequently Tyr2, Ile3 and Gln4 bound in that order. The aromatic ring of Tyr2 formed a π-cation interaction with Cu2+. Oxytocin copper complex structure revealed by paramagnetic relaxation enhancement NMR analyses.

Sulfation Patterns of Saccharides and Heavy Metal Ion Binding.

Sulfated saccharides are an essential part of extracellular matrices, and they are involved in a large number of interactions. Sulfated saccharide matrices in organisms accumulate heavy metal ions in addition to other essential metal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severe and irreversible damage. The effect of the sulfation pattern of saccharides on heavy metal binding preferences is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because standard analytical techniques cannot be used to quantify these interactions. We developed a new strategy that combines enzymatic and chemical synthesis with surface chemistry and label-free electrochemical sensing to study the interactions between well-defined sulfated saccharides and heavy metal ions. By using these tools we showed that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences.

ITO Work Function Tunability by Polarizable Chromophore Monolayers.

The ability to tune the electronic properties of oxide-bearing semiconductors such as Si/SiO2 or transparent metal oxides such as indium-tin oxide (ITO) is of great importance in both electronic and optoelectronic device applications. In this work, we describe a process that was conducted on n-type Si/SiO2 and ITO to induce changes in the substrate work function (WF). The substrates were modified by a two-step synthesis comprising a covalent attachment of coupling agents' monolayer followed by in situ anchoring reactions of polarizable chromophores. The coupling agents and chromophores were chosen with opposite dipole orientations, which enabled the tunability of the substrates' WF. In the first step, two coupling agents with opposite molecular dipole were assembled. The coupling agent with a negative dipole induced a decrease in WF of modified substrates, while the coupling agent with a positive dipole produced an increase in WFs of both ITO and Si substrates. The second modification step consisted of in situ anchoring reaction of polarizable chromophores with opposite dipoles to the coupling layer. This modification led to an additional change in the WFs of both Si/SiO2 and ITO substrates. The WF was measured by contact potential difference and modeled by density functional theory-based theoretical calculations of the WF for each of the assembly steps. A good fit was obtained between the calculated and experimental trends. This ability to design and tune the WF of ITO substrates was implemented in an organic electronic device with improved I- V characteristics in comparison to a bare ITO-based device.

Direct Assembly and Metal-Ion Binding Properties of Oxytocin Monolayer on Gold Surfaces.

Peptides are very common recognition entities that are usually attached to surfaces using multistep processes. These processes require modification of the native peptides and of the substrates. Using functional groups in native peptides for their assembly on surfaces without affecting their biological activity can facilitate the preparation of biosensors. Herein, we present a simple single-step formation of native oxytocin monolayer on gold surface. These surfaces were characterized by atomic force spectroscopy, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy. We took advantage of the native disulfide bridge of the oxytocin for anchoring the peptide to the Au surface, while preserving the metal-ion binding properties. Self-assembled oxytocin monolayer was used by electrochemical impedance spectroscopy for metal-ion sensing leading to subnanomolar sensitivities for zinc or copper ions.

Detection of Cu2+ Ions with GGH Peptide Realized with Si-Nanoribbon ISFET.

The presence of heavy metal ions such as copper in the human body at certain concentrations and specific conditions can lead to the development of different diseases. The currently available analytical detection methods remain expensive, time-consuming, and often require sample pre-treatment. The development of specific and quantitative, easy-in-operation, and cost-effective devices, capable of monitoring the level of Cu2+ ions in environmental and physiological media, is necessary. We use silicon nanoribbon (SiNR) ion-sensitive field effect transistor (ISFET) devices modified with a Gly-Gly-His peptide for the detection of copper ions in a large concentration range. The specific binding of copper ions causes a conformational change of the ligand, and a deprotonation of secondary amine groups. By performing differential measurements, we gain a deeper insight into the details of the ion-ligand interaction. We highlight in particular the importance of considering non-specific interactions to explain the sensors' response.

Diversification of Device Platforms by Molecular Layers: Hybrid Sensing Platforms, Monolayer Doping, and Modeling.

Inorganic materials such as semiconductors, oxides, and metals are ubiquitous in a wide range of device technologies owing to the outstanding robustness and mature processing technologies available for such materials. However, while the important contribution of inorganic materials to the advancement of device technologies has been well established for decades, organic-inorganic hybrid device systems, which merge molecular functionalities with inorganic platforms, represent a newer domain that is rapidly evolving at an increasing pace. Such devices benefit from the great versatility and flexibility of the organic building blocks merged with the robustness of the inorganic platforms. Given the overwhelming wealth of literature covering various approaches for modifying and using inorganic devices, this feature article selectively highlights some of the advances made in the context of the diversification of devices by surface chemistry. Particular attention is given to oxide-semiconductor systems and metallic surfaces modified with organic monolayers. The inorganic device components, such as semiconductors, metals, and oxides, are modified by organic monolayers, which may serve as either active, static, or sacrificial components. We portray research directions within the broader field of organic-inorganic hybrid device systems that can be viewed as specific examples of the potential of such hybrid device systems given their comprehensive capabilities of design and diversification. Monolayer doping techniques where sacrificial organic monolayers are introduced into semiconducting elements are reviewed as a specific case, together with associated requirements for nanosystems, devices, and sensors for controlling doping levels and doping profiles on the nanometric scale. Another series of examples of the flexibility provided by the marriage of organic functional monolayers and inorganic device components are represented by a new class of biosensors, where the organic layer functionality is exploited in a functioning device for sensing. Considerations for relying on oxide-terminated semiconductors rather than the pristine semiconductor material as a platform both for processing and sensing are discussed. Finally, we cover aspects related to the use of various theoretical and computational approaches to model organic-inorganic systems. The main objectives of the topics covered here are (i) to present the advances made in each respective domain and (ii) to provide a comprehensive view of the potential uses of organic monolayers and self-assembly processes in the rapidly evolving field of molecular-inorganic hybrid device platforms and processing methodologies. The directions highlighted here provide a perspective on a future, not yet fully realized, integrated approach where organic monolayers are combined with inorganic platforms in order to obtain versatile, robust, and flexible systems with enhanced capabilities.

Carbon Nanotube and Semiconductor Nanorods Hybrids: Preparation, Characterization, and Evaluation of Photocurrent Generation.

Carbon nanotubes (CNTs) and semiconductor nanocrystals (SCNCs) are known to be interesting donor-acceptor partners due to their unique optical and electronic properties. These exciting features have led to the development of novel composites based on these two nanomaterials and to their characterization for use in various applications, such as components in sensors, transistors, solar cells and biomedical devices. Two approaches based on covalent and noncovalent methods have been suggested for coupling the SCNCs to CNTs. Most covalent conjugation methods used so far were found to disrupt the electronic structure of the CNTs or interfere with charge transfer in the CNT-SCNC interface. Moreover, it offers random and poorly organized nanoparticle coatings. Therefore, noncovalent methods are considered to be ideal for better electronic coupling. However, a key common drawback of noncovalent methods is the lack of stability which hampers their applicability. In this article, a method has been developed to couple semiconductor seeded nanorods onto CNTs through π-π interactions. The CNTs and pyrene conjugated SCNC hybrid materials were characterized by both microscopic and spectroscopic techniques. Fluorescence and photocurrent measurements suggest the proposed pi-stacking approach results in a strong electronic coupling between the CNTs and the SCNCs leading to better photocurrent efficiency than that of a covalent conjugation method reported using similar SCNC material. Overall, the CNT-SCNC films reported in the present study open the scope for the fabrication of optoelectronic devices for various applications.

A highly sensitive square wave voltammetry based biosensor for kinase activity measurements.

An electrochemical biosensor has been developed for ultrasensitive, label-free determination of protein kinase activity. The sensor is composed of a unique peptide monolayer on a gold electrode. It identifies the order change in the monolayer upon phosphorylation, via square wave voltametry (SWV) measurements. Disorder caused by the introduction of the phosphate groups onto the middle of the peptide sequence results in pinhole formation and therefore an increase in the electrochemical signal. The measured sensitivity was 100 nM of kinase and the dynamic range was 100 nM up to 11 µM. Sensitivity was an order of magnitude higher, and the dynamic range wider by two orders of magnitude, as compared to our previously reported impedimetric method, in which the sensitivity was 1 µM, and the dynamic range was 1-20 µM.

Blue-violet photoluminescence of 4-isopropyl-pyridine hydroxide crystals.

There is continuing interest in determining essential structural features of polymer gels, which display photoelectric and/or thermoelectric behavior. One such gel is the blend, poly(4-vinylpyridine-co-butyl methacrylate)/poly(4-vinylpyridine), dissolved in liquid pyridine. Following extended aeration of a three-component mixture, which serves as a model for the gel side chain interactions, crystallization of a new molecule, 4-isopropylpyridine hydroxide (IPPOH), occurs. X-ray diffraction, DFT modeling, and spectroscopy were used to determine the structural, electronic, and luminescent properties of the crystal. The crystal structure reveals molecules forming head-to-tail, hydrogen-bonded chains without base stacking or marked interchain interaction. The molecular chains are characterized by moderately long-lived, blue-violet luminescence excited in the near-UV. Because these photoluminescent properties resemble those of the gel from which the crystals are derived, we may posit similar structural features in the gel for which direct structural analysis is not available.

Biocatalysis versus Molecular Recognition in Sialoside-Selective Neuraminidase Biosensing.

Sialic acid recognition and hydrolysis are essential parts of cellular function and pathogen infectivity. Neuraminidases are enzymes that detach sialic acid from sialosides, and their inhibition is a prime target for viral infection treatment. The connectivity and type of sialic acid influence the recognition and hydrolysis activity of the many different neuraminidases. The common strategies to evaluate neuraminidase activity, recognition, and inhibition rely on extensive labeling and require a large amount of sialylated glycans. The above limitations make the effort of finding viral inhibitors extremely difficult. We used synthetic sialylated glycans and developed a label-free electrochemical method to show that sialoside structural features lead to selective neuraminidase biosensing. We compared Neu5Ac to Neu5Gc sialosides to evaluate the organism-dependent neuraminidase selectivity-sensitivity relationship. We demonstrated that the type of surface and the glycan monolayer density direct the response to either binding or enzymatic activity. We proved that while the hydrophobic glassy carbon surface increases the interaction with the enzyme hydrophobic interface, the negatively charged interface of the lipoic acid monolayer on gold repels the protein and enables biocatalysis. We showed that the sialoside monolayers can serve as tools to evaluate the inhibition of neuraminidases both by biocatalysis and molecular recognition.

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.

Monitoring selectivity in kinase-promoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection.

This paper describes remarkably high sensitivities in the label-free detection of kinase-promoted phosphorylation for 14 different peptide substrates on electrode-immobilized monolayers (gold or nitride) using serine/threonine kinases PKA, PKC, and CaMK2. Peptide substrates were preselected using (33)P-labeling in a microarray of 1024 substrates. The three most active peptides (A1-A3, C1-C3, and M1-M3) were investigated using electrochemical impedance spectroscopy (EIS) and ion-sensitive field effect transistors (ISFETs). Some of the peptide substrates, for example, the PKC-specific substrate PPRRSSIRNAH (C1), showed a remarkably high sensitivity in the EIS-based sensor measurements. Our studies revealed that this high sensitivity is primarily due to the monolayer's packing density. Nanoscopic studies demonstrated a distinct disordering of the C1-monolayer upon phosphorylation, while phosphatase-promoted dephosphorylation regenerated the highly ordered peptide monolayer. As a matter of fact, the initial surface packing of the peptide monolayer mainly determined the level of sensitivity, whereas electrostatic repulsion of the redox-active species was found to be much less important.