Publications

2022
Ariel Shitrit, Sandhya Mardhekar, Israel Alshanski, Prashant jain, Rakesh Raigawali, Chethan D Shanthamurthy, Raghavendra Kikkeri, yitzchaikshlomo, and Mattan Hurevich. 8/19/2022. “Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques.” Chemistry – A European Journal, 28, Pp. e202202193. Abstract

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


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Dana Grunhaus, Estefanía Rossich Molina, Roni Cohen, Tamar Stein, Assaf Friedler, and Mattan Hurevich. 7/12/2022. “Accelerated Multiphosphorylated Peptide Synthesis.” Organic Process Research & Development, 26, 8, Pp. 2492-2497. Abstract

Preparing phosphorylated peptides with multiple adjacent phosphorylations is synthetically difficult, leads to β-elimination, results in low yields, and is extremely slow. We combined synthetic chemical methodologies with computational studies and engineering approaches to develop a strategy that takes advantage of fast stirring, high temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to produce multiphosphorylated peptides at an extremely rapid time and high purity.

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Expeditious Synthesis of a Glycopeptide Library
Dror Ben Abba Amiel and Mattan Hurevich. 7/5/2022. “Expeditious Synthesis of a Glycopeptide Library.” European Journal of Organic Chemistry, e202200623. Abstract

Short proteoglycan fragments are of great importance for biochemical research. The solid-phase synthesis of such glycopeptides relies on excessive use of glycosylated amino acids, extended reaction times, and additional post-assembly deprotection protocols. We employed high-shear mixing for expedient and equimolar O-glycopeptide assembly. We further developed a stirring-based deprotection on the solid support, thus completing the synthesis of a glycopeptide library in a minimal amount of time and purification hurdles. Publisher's Version

 

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Poriah Strauss, Francesca Nuti, Michael Quagliata, Anna Maria Papini, and Mattan Hurevich. 2022. “Accelerated solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites.” Org. Biomol. Chem., Pp. -. Publisher's Version Abstract

Peptide fragments of glycoproteins containing multiple N-glycosylated sites are essential biochemical tools not only to investigate protein–protein interactions but also to develop glycopeptide-based diagnostics and immunotherapy. However, solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites is hampered by difficult couplings, which results in a substantial drop in yield. To increase the final yield, large amounts of reagents but also time-consuming steps are required. Therefore, we propose herein to utilize heating and stirring in combination with low-loading solid supports to set up an accelerated route to obtain, by an efficient High-Temperature Fast Stirring Peptide Synthesis (HTFS-PS), glycopeptides containing multiple N-glycosylated sites using equimolar excess of the precious glycosylated building blocks.

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Effect of Interfacial Properties on Impedimetric Biosensing of the Sialylation Process with a Biantennary N-Glycan-Based Monolayer
Israel Alshanski, Ariel Shitrit, Yonatan Sukhran, Carlo Unverzagt, Mattan Hurevich, and Shlomo Yitzchaik. 2022. “Effect of Interfacial Properties on Impedimetric Biosensing of the Sialylation Process with a Biantennary N-Glycan-Based Monolayer.” LANGMUIR, 38, 2, Pp. 849-855. Abstract

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. Publisher's Version

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Stirring Peptide Synthesis to a New Level of Efficiency
Johnny N. Naoum, Israel Alshanski, Guy Mayer, Poriah Strauss, and Mattan Hurevich. 2022. “Stirring Peptide Synthesis to a New Level of Efficiency.” ORGANIC PROCESS RESEARCH & DEVELOPMENT, 26, 1, Pp. 129-136. Abstract

Accelerating solid-phase synthesis is crucial for accessing a large number of peptides in a short time. Since standard peptide synthesis is usually done under poor diffusion conditions with slow or no mixing of the solid support, acceleration of the process is achieved by applying a large excess of reagents. In this work, overhead stirring and heating were combined to provide accelerated solid-phase peptide synthesis without using an excess of reagent. A new setup that allows both heating and fast stirring was designed specifically for research laboratory-scale peptide synthesis. By increasing the diffusion of both reagents and beads in a narrow dimension reactor, solid-phase reactions were done in seconds and medium-size peptides were synthesized in minutes. Publisher's Version

 

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Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding
Francesco Brunori, Deepak Kumar Padhi, Israel Alshanski, Joanna Freyse, Jan-Niklas Duerig, Anja Penk, Luigi Vaccaro, Mattan Hurevich, Joerg Rademann, and Shlomo Yitzchaik. 2022. “Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.” CHEMBIOCHEM, 23, 3. Abstract

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. Publisher's Version

 

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Translating Solution to Solid Phase Glycosylation Conditions
Yasmeen Bakhatan, Dror Ben Abba Amiel, Yonatan Sukhran, Chieh-Kai Chan, Wei-Chih Lo, Po-Wei Lu, Pin-Hsuan Liao, Cheng-Chung Wang, and Mattan Hurevich. 2022. “Translating Solution to Solid Phase Glycosylation Conditions.” Chem. Commun., 58, Pp. 11256-11259. Publisher's Version Abstract

Optimizing glycosylation conditions for automated glycan assembly is highly challenging, demand wasteful use of precious building blocks and rely on nontrivial analyses. We developed a semi-quantitative method for automated optimization of glycosylation temperature that utilized minute quantities of donors and translated those conditions to solid-phase glycan assembly.

 

2021
D. Ben Abba Amiel, I. Alshanski, and M. Hurevich. 2021. “2.17 - Automated Oligosaccharide Synthesis: Development of the Glyconeer®.” In Comprehensive Glycoscience (Second Edition), edited by Joseph J. Barchi, Second Edition, Pp. 548-560. Oxford: Elsevier. Abstract

Developing automated platforms is essential for accelerating the preparation of bioactive compound libraries. After a decade of biopolymer synthesis, it is clear that automating these processes was pivotal to the development of biochemistry and chemical biology for advancing medicinal chemistry and for providing new diagnoses and therapeutic tools. Synthesis of glycans and glycoconjugates is far more complicated than the other biopolymer families. Automating glycan synthesis has been a long and hard journey which is still ongoing. This chapter focuses on the development of a commercial platform, Glyconeer™, aimed to automate the synthesis of glycans. The design and process leading to the establishment of the current setup are described. The unique considerations required from a system that is suitable for glycan synthesis are the focus of the chapter. We explain how the selected setup architecture and its unique features comply with the unusual demands of automated solid phase synthesis of glycans. We will present the common modules, building blocks, and chemistries used by the synthesizer. Preparation, handling, and running of both software and hardware are presented from the user point of view. A critical view of the limitations and advantages of the system is aimed to provide a roadmap for future improvement. Publisher's Version

 

 

Automated Synthesis of Heavily Phosphorylated Peptides
Dana Grunhaus, Assaf Friedler, and Mattan Hurevich. 2021. “Automated Synthesis of Heavily Phosphorylated Peptides.” EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, 2021, 26, Pp. 3737-3742. Abstract

Multi phosphorylated peptides are key tools in understanding the biological roles of protein phosphorylation patterns. In this work, we focused on multi phosphorylated peptides with over four, clustered, phosphorylation sites that are termed herein heavily phosphorylated peptides (HPPs). The synthesis of heavily phosphorylated peptides is extremely difficult and requires the use of a wide temperature range. Standard peptide synthesizers are incapable of both cooling and heating, which impedes the automated synthesis of those peptides. Herein, we used the oligosaccharide synthesizer Glyconeer 2.1 to develop a protocol for the automated synthesis of heavily phosphorylated peptides. The Glyconeer 2.1 is able to both cool and heat, which enabled the development of highly controlled coupling and deprotection conditions that were used for the automated synthesis of four different heavily phosphorylated peptides with five or more, clustered, phosphorylation sites. Our approach paves the way for an easy automated synthesis of a variety of heavily phosphorylated peptides. Publisher's Version

 

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Determining the structure and binding mechanism of oxytocin-Cu2+ complex using paramagnetic relaxation enhancement NMR analysis
Israel Alshanski, Deborah E. Shalev, Shlomo Yitzchaik, and Mattan Hurevich. 2021. “Determining the structure and binding mechanism of oxytocin-Cu2+ complex using paramagnetic relaxation enhancement NMR analysis.” JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY, 26, 7, Pp. 809-815. Abstract

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 pi-cation interaction with Cu2+Y. Publisher's Version

 

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Electrochemical biosensing platform based on complex biantennary N-glycan for detecting enzymatic sialylation processes
Israel Alshanski, Yonatan Sukhran, Evgeniy Mervinetsky, Carlo Unverzagt, Shlomo Yitzchaik, and Mattan Hurevich. 2021. “Electrochemical biosensing platform based on complex biantennary N-glycan for detecting enzymatic sialylation processes.” BIOSENSORS & BIOELECTRONICS, 172. Abstract

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. Publisher's Version

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The influence of surface proximity on photoswitching activity of stilbene-functionalized N-heterocyclic carbene monolayers
Shahar Dery, Israel Alshanski, Evgeniy Mervinetsky, Daniel Feferman, Shlomo Yitzchaik, Mattan Hurevich, and Elad Gross. 2021. “The influence of surface proximity on photoswitching activity of stilbene-functionalized N-heterocyclic carbene monolayers.” CHEMICAL COMMUNICATIONS, 57, 51, Pp. 6233-6236. Abstract

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. Publisher's Version

 

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Non-covalently embedded oxytocin in alkanethiol monolayer as Zn2+ selective biosensor
Jessica Attia, Sivan Nir, Evgeniy Mervinetsky, Dora Balogh, Agata Gitlin-Domagalska, Israel Alshanski, Meital Reches, Mattan Hurevich, and Shlomo Yitzchaik. 2021. “Non-covalently embedded oxytocin in alkanethiol monolayer as Zn2+ selective biosensor.” SCIENTIFIC REPORTS, 11, 1. Abstract

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. Publisher's Version

 

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Xiaoqian Li, Zhi Ma, Rongkun Liu, Mattan Hurevich, and You Yang. 2021. “Photolabile Protecting Group-Mediated Synthesis of 2-Deoxy-Glycosides.” CHINESE JOURNAL OF CHEMISTRY, 39, 12, Pp. 3309-3314. Abstract

Main observation and conclusion A green and efficient photolabile protecting group (PPG)-mediated glycosidation approach for the synthesis of 2-deoxy-glycosides is reported. By employing ortho-nitrobenzyl carbonate (oNBC) as PPG, N,N-dimethylformamide (DMF)-modulated SPhosAuNTf(2)-promoted glycosidation with per-oNBC-protected 2-deoxy-glycosyl ynenoates affords the 2-deoxy-glycosides with moderate to excellent alpha-selectivities presumably depending on the reactivities of the acceptor alcohols. Based on the PPG-mediated glycosidation approach, oligosaccharides with three to six oNBC groups are effectively achieved. The multiple oNBC groups in the 2-deoxy-glycosides are completely cleaved by irradiation at 365 nm, resulting in the desired 2-deoxy-glycosides in an efficient manner without affecting the common alkyne, alkene, azide functional groups and the traditional protecting groups on the aglycones. Publisher's Version

 

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2020
The breaking beads approach for photocleavage from solid support
Yasmeen Bakhatan, Israel Alshanski, Dana Grunhaus, and Mattan Hurevich. 2020. “The breaking beads approach for photocleavage from solid support.” ORGANIC & BIOMOLECULAR CHEMISTRY, 18, 22, Pp. 4183-4188. Abstract

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. Publisher's Version

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Cyclizing Painkillers: Development of Backbone-Cyclic TAPS Analogs
Alaa Talhami, Avi Swed, Shmuel Hess, Oded Ovadia, Sarit Greenberg, Adi Schumacher-Klinger, David Rosenthal, Deborah E. Shalev, Mattan Hurevich, Philip Lazarovici, Amnon Hoffman, and Chaim Gilon. 2020. “Cyclizing Painkillers: Development of Backbone-Cyclic TAPS Analogs.” FRONTIERS IN CHEMISTRY, 8. Abstract

Painkillers are commonly used medications. Native peptide painkillers suffer from various pharmacological disadvantages, while small molecule painkillers like morphine are highly addictive. We present a general approach aimed to use backbone-cyclization to develop a peptidomimetic painkiller. Backbone-cyclization was applied to transform the linear peptide Tyr-Arg-Phe-Sar (TAPS) into an active backbone-cyclic peptide with improved drug properties. We designed and synthesized a focused backbone-cyclic TAPS library with conformational diversity, in which the members of the library have the generic name TAPS c(n-m) where n and m represent the lengths of the alkyl chains on the nitrogens of Gly and Arg, respectively. We used a combined screening approach to evaluate the pharmacological properties and the potency of the TAPS c(n-m) library. We focused on an in vivo active compound, TAPS c(2-6), which is metabolically stable and has the potential to become a peripheral painkiller being a full mu opioid receptor functional agonist. To prepare a large quantity of TAPS c(2-6), we optimized the conditions of the on-resin reductive alkylation step to increase the efficiency of its SPPS. NMR was used to determine the solution conformation of the peptide lead TAPS c(2-6). Publisher's Version

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Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms
Mamidi Samarasimhareddy, Guy Mayer, Mattan Hurevich, and Assaf Friedler. 2020. “Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms.” ORGANIC & BIOMOLECULAR CHEMISTRY, 18, 18, Pp. 3405-3422. Abstract

Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described. Publisher's Version

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A Rapid and Efficient Building Block Approach for Click Cyclization of Peptoids
Mamidi Samarasimhareddy, Mai Shamir, Deborah E. Shalev, Mattan Hurevich, and Assaf Friedler. 2020. “A Rapid and Efficient Building Block Approach for Click Cyclization of Peptoids.” FRONTIERS IN CHEMISTRY, 8. Abstract

Cyclic peptide-peptoid hybrids possess improved stability and selectivity over linear peptides and are thus better drug candidates. However, their synthesis is far from trivial and is usually difficult to automate. Here we describe a new rapid and efficient approach for the synthesis of click-based cyclic peptide-peptoid hybrids. Our methodology is based on a combination between easily synthesized building blocks, automated microwave assisted solid phase synthesis and bioorthogonal click cyclization. We proved the concept of this method using the INS peptide, which we have previously shown to activate the HIV-1 integrase enzyme. This strategy enabled the rapid synthesis and biophysical evaluation of a library of cyclic peptide-peptoid hybrids derived from HIV-1 integrase in high yield and purity. The new cyclic hybrids showed improved biological activity and were significantly more stable than the original linear INS peptide. Publisher's Version

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On Sensing Principles Using Temporally Extended Bar Codes
Vasileios Athanasiou, Kiran Kumar Tadi, Mattan Hurevich, Shlomo Yitzchaik, Aldo Jesorka, and Zoran Konkoli. 2020. “On Sensing Principles Using Temporally Extended Bar Codes.” IEEE SENSORS JOURNAL, 20, 13, Pp. 6782-6791. Abstract

The detection of ionic variation patterns could be a significant marker for the diagnosis of neurological and other diseases. This paper introduces a novel idea for training chemical sensors to recognise patterns of ionic variations. By using an external voltage signal, a sensor can be trained to output distinct time-series signals depending on the state of the ionic solution. Those sequences can be analysed by a relatively simple readout layer for diagnostic purposes. The idea is demonstrated on a chemical sensor that is sensitive to zinc ions with a simple goal of classifying zinc ionic variations as either stable or varying. The study features both theoretical and experimental results. By extensive numerical simulations, it has been shown that the proposed method works successfully in silico. Distinct time-series signals are found which occur with a high probability under only one class of ionic variations. The related experimental results point in the right direction. Publisher's Version

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