Glycosylation is among the most common post-translational modifications of proteins. There is a great synthetic and practical difficulty in the assembly and deprotection of glycopeptides. State-of-the-art methods for glycopeptide synthesis are wasteful of glycosylated amino acids, are slow, and suffer from low yields. These shortcomings hamper accessibility to multiply glycosylated peptides. We report the accelerated, high-shear stirring-assisted synthesis of multiply O-glycosylated peptides. The equimolar assembly was streamlined with deacetylation to provide multiglycosylated peptides at high purity. Cadherin-derived multiglycosylated peptides synthesized in large quantities provided selective Listeria monocytogenes electrochemical biosensing.

Semiconducting single-walled carbon nanotubes (sc-SWCNTs) are of great potential for vapor sensing. However, sc-SWCNTs lack recognition features for discriminating between sparsely functionalized moieties, molecules with similar structural features, and enantiomer pairs. This becomes a major setback in discriminating between volatile organic compounds (VOCs). Here, we used two galactosides decorated with aromatic groups as a recognition layer in chemoresistive sc-SWCNT sensors to produce chiral preference toward six terpenoid enantiomers. The multichirality and multifunctionality of a monosaccharide scaffold were exploited to maximize the limited interacting features associated with VOCs. The developed system establishes a robust and tunable platform for enantioselective gas sensing.

Cholera is a severe infectious disease caused by Vibrio cholerae. The disease primarily spreads through contaminated food and water sources; it remains a significant global health concern. The pathogenesis of Vibrio cholerae is facilitated by the secreted neuraminidase, Vibrio cholerae neuraminidase (VCNA). This neuraminidase cleaves host cell surface sialic acids, which leads to bacterial colonization and infection progression. This study presents the development of a label-free VCNA biosensor based on electrochemical impedance spectroscopy. The biosensor relies on synthetic sialosides that form self-assembled monolayers on gold electrodes. The system demonstrated selective detection of VCNA activity through distinct impedance variations corresponding to the enzymatic cleavage of the sialoside substrates. The VCNA activity was evaluated under varying environmental conditions, including different media and pH values. This approach provides insights into developing robust biosensing platforms for bacterial detection, offering potential applications in various diagnostic and monitoring systems.

Heparan sulfate (HS) interactions with interleukin 8 (IL-8) are crucial for immune system response. The structural features of the HS and the environmental entities, such as metal ions, can regulate these interactions. However, it is challenging to evaluate the effect of each parameter on the interactions because of low accessibility to well-defined saccharides and the lack of characteristic features to be determined by analytical tools. We evaluated the effect of the HS structural features on IL-8 binding affinity utilizing electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). We showed that the metal ions Ca²⁺ and Mg²⁺ dissimilarly mediate the interactions of HS and IL-8 in structure-dependent manner of the HS. We showed that in all glycans, a positive synergistic effect on IL-8 binding was observed. For several glycans, the presence of ions resulted in a dramatic increase in the affinity to IL-8, while for other glycans, a milder effect was observed. This demonstrated that both structural motifs and environmental features are crucial for maintaining the interactions between the HS and IL-8.

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.

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.

Streptococcus pneumoniae is a pathogenic bacterium that contains the surface-bound neuraminidase, NanA. NanA has two domains that interact with sialosides. It is hard to determine the contribution of each domain separately on catalysis or binding. In this work, we used biochemical methods to obtain the separated domains, applied electrochemical and surface analysis approaches, and determined the catalytic and binding preferences toward a surface-bound library of sialosides. Impedimetric studies on two different surfaces revealed that protein–surface interactions provide a tool for distinguishing the unique contribution of each domain at the interface affecting the substrate preference of the enzyme in different surroundings. We showed that each domain has a sialoside-specific affinity. Furthermore, while the interaction of the sialoside-covered surface with the carbohydrate-binding domain results in an increase in impedance and binding, the catalytic domain adheres to the surface at high concentrations but retains its catalytic activity at low concentrations.

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 (R_CT) caused due to the binding of bacteria to RPMs. Pseudomonas aeruginosa was found to cause the most prominent increase in R_CT 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.

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.

 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. 

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.

 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+. 

 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. 

 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.

 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.

 Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. We report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer as a proof-of-concept, three substrate peptides selected from constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) were designed to demonstrate the feasibility. The assay showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2 ± 0.2 fmol μg-1 min-1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, suggesting different endogenous kinases for each substrate peptide. Without the conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 h. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. With the ease of design, sensitivity, accuracy, and reproducibility, this approach may offer rapid and sensitive assays for targeted quantification of the multiplexed substrate-specific kinase activity of small amounts of sample. 

 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. 

  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.