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.

Accelerated Solid Phase Glycan Synthesis: ASGS.

Solid phase synthesis is the most dominant approach for the preparation of biological oligomers as it enables the introduction of monomers iteratively. Accelerated solid phase synthesis of biological oligomers is crucial for chemical biology, but its application to the synthesis of oligosaccharides is not trivial. Solid-phase oligosaccharide assembly is a slow process performed in a variety of conditions and temperatures, requires an inert gas atmosphere, and demands high excess of glycosyl donors. The process is done in special synthesizers and poor mixing of the solid support increases the risk of diffusion-independent hydrolysis of the activated donors. High shear stirring is a new way to accelerate solid phase synthesis. The efficient mixing ensures that reactive intermediates can diffuse faster to the solid support thereby increasing the kinetics of the reactions. We report here a stirring-based accelerated solid-phase oligosaccharide synthesis. We harnessed high shear mixing to perform diffusion-dependent glycosylation in a short reaction time. We minimized the use of glycosyl donors and the need to use an inert atmosphere. We showed that by tailoring the deprotection and glycosylation conditions to the same temperature, assembly steps are performed continuously, and full glycosylation cycles are completed in minutes.

Unexpected Nucleophile Masking in Acyl Transfer to Sterically Crowded and Conformationally Restricted Galactosides.

Design and synthesis of orthogonally protected monosaccharide building blocks are crucial for the preparation of well-defined oligosaccharides in a stereo- and regiocontrolled manner. Selective introduction of protecting groups to partially protected monosaccharides is nontrivial due to the often unpredictable electronic, steric, and conformational effects of the substituents. Abolished reactivity toward a commonly used Lewis base-catalyzed acylation of O-2 was observed in conformationally restricted 4,6-O-benzylidene-3-O-Nap galactoside. Investigation of analogous systems, crystallographic characterization, and quantum chemical calculations highlighted the overlooked conformational and steric considerations, the combination of which produces a unique passivity of the 2-OH nucleophile. Evaluating the role of electrophile counterion and auxiliary base in the acylation of the sterically crowded and conformationally restricted galactoside system revealed an alternative Brønsted base-driven reaction pathway via nucleophilic activation. Insights gained from this model system were utilized to access the target galactoside intermediate within the envisioned synthetic route. The acylation strategy described herein can be implemented in future syntheses of key monomeric building blocks with unique protecting group hierarchies.

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.

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.

The breaking beads approach for photocleavage from solid support.

Photocleavage from polystyrene beads is a pivotal reaction for solid phase synthesis that relies on photolabile linkers. Photocleavage from intact porous polystyrene beads is not optimal because light cannot penetrate into the beads and the surface area exposed to irradiation is limited. Thus, hazardous, technically challenging and expensive setups are used for photocleavage from intact beads. We developed a new concept in which grinding the beads during or prior to irradiation is employed as an essential part of the photocleavage process. By grinding the beads we are exposing more surface area to the light source, hence, photocleavage can be performed even using a simple benchtop LED setup. This approach proved very efficient for photocleavage of various model compounds including fully protected oligosaccharides.

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.

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.

The influence of surface proximity on photoswitching activity of stilbene-functionalized N-heterocyclic carbene monolayers.

Self-assembly of photo-responsive molecules is a robust technology for reversibly tuning the properties of functional materials. Herein, we probed the crucial role of surface-adsorbate interactions on the adsorption geometry of stilbene-functionalized N-heterocyclic carbenes (stilbene-NHCs) monolayers and its impact on surface potential. Stilbene-NHCs on Au film accumulated in a vertical orientation that enabled high photoisomerization efficiency and reversible changes in surface potential. Strong metal-adsorbate interactions led to flat-lying adsorption geometry of stilbene-NHCs on Pt film, which quenched the photo-isomerization influence on surface potential. It is identified that photo-induced response can be optimized by positioning the photo-active group in proximity to weakly-interacting surfaces.

Electrochemical biosensing platform based on complex biantennary N-glycan for detecting enzymatic sialylation processes.

Sialylated glycans and glycoproteins are involved in cellular communication and are crucial for distinguishing between signal pathways. Sialylation levels and patterns modulate recognition events and are regulated by the enzymatic activity of sialyltransferases and neuraminidases. Abnormal activity of these enzymes is related to diseases such as cancer and viral infection. Monitoring these enzymatic activities offers valuable diagnostic tools. This work presents an impedimetric biosensing platform for following and detecting sialylation and desialylation processes. This platform is based on a native biantennary N-glycan substrate attached to a glassy carbon electrode. Changes in the molecular layer, as a result of enzymatic reactions, were detected by electrochemical impedance spectroscopy, displaying high sensitivity to the enzymatic surface reactions. Increase in the molecular layer roughness in response to the sialylation was visualized using atomic force microscopy. After enzymatic sialylation, the presence of sialic acid was confirmed using cyclic voltammetry by coupling of the redox active marker aminoferrocene. The sialylation showed selectivity toward the N-glycan compared to another glycan substrate. A time dependent sialylation was followed by electrochemical impedance spectroscopy, proving that the new system can be applied to evaluate the enzymatic kinetics. Our findings suggest that analyzing sialylation processes using this platform can become a useful tool for the detection of pathological states and pathogen invasion.

Non-covalently embedded oxytocin in alkanethiol monolayer as Zn2+ selective biosensor.

Peptides are commonly used as biosensors for analytes such as metal ions as they have natural binding preferences. In our previous peptide-based impedimetric metal ion biosensors, a monolayer of the peptide was anchored covalently to the electrode. Binding of metal ions resulted in a conformational change of the oxytocin peptide in the monolayer, which was measured using electrochemical impedance spectroscopy. Here, we demonstrate that sensing can be achieved also when the oxytocin is non-covalently integrated into an alkanethiol host monolayer. We show that ion-binding cause morphological changes to the dense host layer, which translates into enhanced impedimetric signals compared to direct covalent assembly strategies. This biosensor proved selective and sensitive for Zn2+ ions in the range of nano- to micro-molar concentrations. This strategy offers an approach to utilize peptide flexibility in monitoring their response to the environment while embedded in a hydrophobic monolayer.

A zinc selective oxytocin based biosensor.

Oxytocin is a peptide hormone with high affinity to both Zn2+ and Cu2+ ions compared to other metal ions. This affinity makes oxytocin an attractive recognition layer for monitoring the levels of these essential ions in biofluids. Native oxytocin cannot differentiate between Cu2+ and Zn2+ ions and hence it is not useful for sensing Zn2+ in the presence of Cu2+. We elucidated the effect of the terminal amine group of oxytocin on the affinity toward Cu2+ using theoretical calculations. We designed a new Zn2+ selective oxytocin-based biosensor that utilizes the terminal amine for surface anchoring, also preventing the response to Cu2+. The biosensor shows exceptional selectivity and very high sensitivity to Zn2+ in impedimetric biosensing. This study shows for the first time an oxytocin derived sensor that can be used directly for sensing Zn2+ in the presence of Cu2+.

Copper Induced Conformational Changes of Tripeptide Monolayer Based Impedimetric Biosensor.

Copper ions play a major role in biological processes. Abnormal Cu2+ ions concentrations are associated with various diseases, hence, can be used as diagnostic target. Monitoring copper ion is currently performed by non-portable, expensive and complicated to use equipment. We present a label free and a highly sensitive electrochemical ion-detecting biosensor based on a Gly-Gly-His tripeptide layer that chelate with Cu2+ ions. The proposed sensing mechanism is that the chelation results in conformational changes in the peptide that forms a denser insulating layer that prevents RedOx species transfer to the surface. This chelation event was monitored using various electrochemical methods and surface chemistry analysis and supported by theoretical calculations. We propose a highly sensitive ion-detection biosensor that can detect Cu2+ ions in the pM range with high SNR parameter.

Oxytocin-Monolayer-Based Impedimetric Biosensor for Zinc and Copper Ions.

Zinc and copper are essential metal ions for numerous biological processes. Their levels are tightly maintained in all body organs. Impairment of the Zn2+ to Cu2+ ratio in serum was found to correlate with many disease states, including immunological and inflammatory disorders. Oxytocin (OT) is a neuropeptide, and its activity is modulated by zinc and copper ion binding. Harnessing the intrinsic properties of OT is one of the attractive ways to develop valuable metal ion sensors. Here, we report for the first time an OT-based metal ion sensor prepared by immobilizing the neuropeptide onto a glassy carbon electrode. The developed impedimetric biosensor was ultrasensitive to Zn2+ and Cu2+ ions at physiological pH and not to other biologically relevant ions. Interestingly, the electrochemical impedance signal of two hemicircle systems was recorded after the attachment of OT to the surface. These two semicircles suggest two capacitive regions that result from two different domains in the OT monolayer. Moreover, the change in the charge-transfer resistance of either Zn2+ or Cu2+ was not similar in response to binding. This suggests that the metal-dependent conformational changes of OT can be translated to distinct impedimetric data. Selective masking of Zn2+ and Cu2+ was used to allow for the simultaneous determination of zinc to copper ions ratio by the OT sensor. The OT sensor was able to distinguish between healthy control and multiple sclerosis patients diluted sera samples by determining the Zn/Cu ratio similar to the state-of-the-art techniques. The OT sensor presented herein is likely to have numerous applications in biomedical research and pave the way to other types of neuropeptide-derived sensors.