N-Heterocyclic Carbene Monolayers on Metal-Oxide Films: Correlations between Adsorption Mode and Surface Functionality.

N-Heterocyclic carbene (NHC) ligands have been self-assembled on various metal and semimetal surfaces, creating a covalent bond with surface metal atoms that led to high thermal and chemical stability of the self-assembled monolayer. This study explores the self-assembly of NHCs on metal-oxide films (CuOx, FeOx, and TiOx) and reveals that the properties of these metal-oxide substrates play a pivotal role in dictating the adsorption behavior of NHCs, influencing the decomposition route of the monolayer and its impact on work function values. While the attachment of NHCs onto CuOx is via coordination with surface oxygen atoms, NHCs interact with TiOx through coordination with surface metal atoms and with FeOx via coordination with both metal and oxygen surface atoms. These distinct binding modes arise due to variances in the electronic properties of the metal atoms within the investigated metal-oxide films. Contact angle and ultraviolet photoelectron spectroscopy measurements have shown a significantly higher impact of F-NHC adsorption on CuOx than on TiOx and FeOx , correlated to a preferred, averaged upright orientation of F-NHC on CuOx.

Selective Deposition of N-Heterocyclic Carbene Monolayers on Designated Au Microelectrodes within an Electrode Array.

The incorporation of organic self-assembled monolayers (SAMs) in microelectronic devices requires precise spatial control over the self-assembly process. In this work, selective deposition of N-heterocyclic carbenes (NHCs) on specific electrodes within a two-microelectrode array is achieved by using pulsed electrodeposition. Spectroscopic analysis of the NHC-coated electrode arrays reveals that each electrode is selectively coated with a designated NHC. The impact of NHC monolayers on the electrodes' work function is quantified using Kelvin probe force microscopy. These measurements demonstrate that the work function values of each electrode can be independently tuned by the adsorption of a specific NHC. The presented deposition method enables to selectively coat designated microelectrodes in an electrode array with chosen NHC monolayers for tuning their chemical and electronic functionality.

N-Heterocyclic Carbene Based Nanolayer for Copper Film Oxidation Mitigation.

The wide use of copper is limited by its rapid oxidation. Main oxidation mitigation approaches involve alloying or surface passivation technologies. However, surface alloying often modifies the physical properties of copper, while surface passivation is characterized by limited thermal and chemical stability. Herein, we demonstrate an electrochemical approach for surface-anchoring of an N-heterocyclic carbene (NHC) nanolayer on a copper electrode by electro-deposition of alkyne-functionalized imidazolium cations. Water reduction reaction generated a high concentration of hydroxide ions that induced deprotonation of imidazolium cations and self-assembly of NHCs on the copper electrode. In addition, alkyne group deprotonation enabled on-surface polymerization by coupling surface-anchored and solvated NHCs, which resulted in a 2 nm thick NHC-nanolayer. Copper film coated with a NHC-nanolayer demonstrated high oxidation resistance at elevated temperatures and under alkaline conditions.

Electrochemical deposition of N-heterocyclic carbene monolayers on metal surfaces.

N-heterocyclic carbenes (NHCs) have been widely utilized for the formation of self-assembled monolayers (SAMs) on various surfaces. The main methodologies for preparation of NHCs-based SAMs either requires inert atmosphere and strong base for deprotonation of imidazolium precursors or the use of specifically-synthesized precursors such as NHC(H)[HCO3] salts or NHC-CO2 adducts. Herein, we demonstrate an electrochemical approach for surface-anchoring of NHCs which overcomes the need for dry environment, addition of exogenous strong base or restricting synthetic steps. In the electrochemical deposition, water reduction reaction is used to generate high concentration of hydroxide ions in proximity to a metal electrode. Imidazolium cations were deprotonated by hydroxide ions, leading to carbenes formation that self-assembled on the electrode's surface. SAMs of NO2-functionalized NHCs and dimethyl-benzimidazole were electrochemically deposited on Au films. SAMs of NHCs were also electrochemically deposited on Pt, Pd and Ag films, demonstrating the wide metal scope of this deposition technique.

Self-Assembled Monolayers of Nitron: Self-Activated and Chemically Addressable N-Heterocyclic Carbene Monolayers with Triazolone Structural Motif.

N-heterocyclic carbenes (NHCs) have emerged as a unique molecular platform for the formation of self-assembled monolayers (SAMs) on various surfaces. However, active carbene formation requires deprotonation of imidazolium salt precursors, which is mostly facilitated by exposure of the salt to exogenous base. Base residues were found to be adsorbed on the metal surface and hindered the formation of well-ordered carbene-based monolayers. Herein, we show that nitron, a triazolone-based compound that freely tautomerizes to a carbene, can spontaneously self-assemble into monolayers on Pt and Au surfaces, which obviates the necessity for base-induced deprotonation for active carbene formation. SAMs of nitron were found to be thermally stable and could not be displaced by thiols, and thus their high chemical stability was demonstrated. The amino group in surface-anchored nitron was shown to be chemically available for SN 2 reactions, and makes surface-anchored nitron a chemically addressable cross-linking reagent for surface modifications.

Atomic force microscopy characterization of kinase-mediated phosphorylation of a peptide monolayer.

We describe the detailed microscopic changes in a peptide monolayer following kinase-mediated phosphorylation. A reversible electrochemical transformation was observed using square wave voltammetry (SWV) in the reversible cycle of peptide phosphorylation by ERK2 followed by dephosphorylation by alkaline phosphatase. A newly developed method for analyzing local roughness, measured by atomic force microscope (AFM), showed a bimodal distribution. This may indicate either a hole-formation mechanism and/or regions on the surface in which the peptide changed its conformation upon phosphorylation, resulting in increased roughness and current. Our results provide the mechanistic basis for developing biosensors for detecting kinase-mediated phosphorylation in disease.

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.

Integrating proteomics with electrochemistry for identifying kinase biomarkers.

We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. Our approach combines phosphoproteomics for high throughput cancer-related biomarker discovery from patient tissues and an impedimetric kinase activity biosensor for sensitive validation. Using non-small-cell lung cancer (NSCLC) as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed 198 phosphoproteins that are over-phosphorylated in NSCLC. Among the differentially regulated phosphorylation sites, the most significant alteration was in residue S165 in the Hepatoma Derived Growth Factor (HDGF) protein. Hence, HDGF was selected as a model system for the electrochemical studies. Further motif-based analysis of this altered phosphorylation site revealed that extracellular-signal-regulated kinase 1/2 (ERK1/2) are most likely to be the corresponding kinases. For validation of the kinase-substrate pair, densely packed peptide monolayers corresponding to the HDGF phosphorylation site were coupled to a gold electrode. Phosphorylation of the monolayer by ERK2 and dephosphorylation by alkaline phosphatase (AP) were detected by electrochemical impedance spectroscopy (EIS) and surface roughness analysis. Compared to other methods for quantification of kinase concentration, this label-free electrochemical assay offers the advantages of ultra-sensitivity as well as higher specificity for the detection of cancer-related kinase-substrate pair. With implementation of multiple kinase-substrate biomarker pairs, we expect this integrated approach to become a high throughput platform for discovery and validation of phosphorylation-mediated biomarkers.

The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site.

Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.

The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes.

In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ∼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar membranes and charged proteins or biopolymers for encapsulation and delivery applications.