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Öğe Electrochemical MIP sensor for butyrylcholinesterase(Mdpi, 2019) Özçelikay, Göksu; Kurbanoğlu, Sevinç; Zhang, Xiaorong; Söz, Çağla; Wollenberger, Ulla; Özkan, Sibel Ayşıl; Scheller, Frieder W.Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range.Öğe Electrochemical MIP sensors for environmental analysis(Springer, 2023) Yarman, Aysu; Kurbanoğlu, Sevinç; Zhang, Xiaorong; Scheller, Frieder W.[Özet yok]Öğe Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?(Elsevier Advanced Technology, 2018) Zhang, Xiaorong; Yarman, Aysu; Erdossy, Julia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J.; Scheller, Frieder W.Molecularly imprinted polymer (MP) nanofilrns for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of similar to 5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered.Öğe Enzymes as tools in MIP-Sensors(Mdpi, 2017) Yarman, Aysu; Jetzschmann, Katharina J.; Neumann, Bettina; Zhang, Xiaorong; Wollenberger, Ulla; Cordin, Aude; Scheller, Frieder W.Molecularly imprinted polymers (MIPs) have the potential to complement antibodies in bioanalysis, are more stable under harsh conditions, and are potentially cheaper to produce. However, the affinity and especially the selectivity of MIPs are in general lower than those of their biological pendants. Enzymes are useful tools for the preparation of MIPs for both low and high-molecular weight targets: As a green alternative to the well-established methods of chemical polymerization, enzyme-initiated polymerization has been introduced and the removal of protein templates by proteases has been successfully applied. Furthermore, MIPs have been coupled with enzymes in order to enhance the analytical performance of biomimetic sensors: Enzymes have been used in MIP-sensors as tracers for the generation and amplification of the measuring signal. In addition, enzymatic pretreatment of an analyte can extend the analyte spectrum and eliminate interferences.Öğe How an ACE2 mimicking epitope-MIP nanofilm recognizes template-related peptides and the receptor binding domain of SARS-CoV-2(Royal Society of Chemistry, 2022) Zhang, Xiaorong; Waffo, Armel T.; Yarman, Aysu; Kovacs, Norbert; Bognar, Zsofia; Wollenberger, Ulla; El-Sherbiny, Ibrahim M.; Hassan, Rabeay Y. A.; Bier, Frank F.; Gyurcsanyi, Robert E; Zebger, Ingo; Scheller, Frieder W.Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a similar to 5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, i.e. implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, i.e. the central Cys488 substituted by a serine, results in ca. 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.Öğe Imprinted Polymers on the Route to Plastibodies for Biomacromolecules (MIPs), Viruses (VIPs), and Cells (CIPs)(Springer, 2024) Yarman, Aysu; Zhang, Xiaorong; Bagheri, Mandien; El-Sherbiny, Ibrahim M.; Hassan, Rabeay Y. A.; Kurbanoğlu, Sevinç; Waffo, Armel Franklin Tadjoung; Zebger, Ingo; Karabulut, Tutku Ceren; Bier, Frank F.; Lieberzeit, Peter; Scheller, Frieder W.Around 30% of the scientific papers published on imprinted polymers describe the recognition of proteins, nucleic acids, viruses, and cells. The straightforward synthesis from only one up to six functional monomers and the simple integration into a sensor are significant advantages as compared with enzymes or antibodies. Furthermore, they can be synthesized against toxic substances and structures of low immunogenicity and allow multi-analyte measurements via multi-template synthesis. The affinity is sufficiently high for protein biomarkers, DNA, viruses, and cells. However, the cross-reactivity of highly abundant proteins is still a challenge.Öğe Insights in electrosynthesis, target binding, and stability of peptide-imprinted polymer nanofilms(2021) Yarman, Aysu; Caserta, Giorgio; Gyurcsányi, Róbert E.; Wollenberger, Ulla; Zhang, Xiaorong; Zebger, Ingo; Supala, Eszter; Scheller, Frieder W.Molecularly imprinted polymer (MIP) nanofilms have been successfully implemented for the recognition of different target molecules: however, the underlying mechanistic details remained vague. This paper provides new insights in the preparation and binding mechanism of electrosynthesized peptide-imprinted polymer nanofilms for selective recognition of the terminal pentapeptides of the ß-chains of human adult hemoglobin, HbA, and its glycated form HbA1c. To differentiate between peptides differing solely in a glucose adduct MIP nanofilms were prepared by a two-step hierarchical electrosynthesis that involves first the chemisorption of a cysteinyl derivative of the pentapeptide followed by electropolymerization of scopoletin. This approach was compared with a random single-step electrosynthesis using scopoletin/pentapeptide mixtures. Electrochemical monitoring of the peptide binding to the MIP nanofilms by means of redox probe gating revealed a superior affinity of the hierarchical approach with a Kd value of 64.6 nM towards the related target. Changes in the electrosynthesized non-imprinted polymer and MIP nanofilms during chemical, electrochemical template removal and rebinding were substantiated in situ by monitoring the characteristic bands of both target peptides and polymer with surface enhanced infrared absorption spectroscopy. This rational approach led to MIPs with excellent selectivity and provided key mechanistic insights with respect to electrosynthesis, rebinding and stability of the formed MIPsÖğe Label-Free MIP sensors for protein biomarkers(Springer, 2017) Zhang, Xiaorong; Jetzschmann, Katharina; Yarman, Aysu; Scheller, Frieder W.; Wollenberger, Ulla[Özet yok]Öğe “Out of pocket” protein binding—A dilemma of epitope imprinted polymers revealed for human hemoglobin(2021) Zhang, Xiaorong; Caserta, Giorgio; Supala, Eszter; Wollenberger, Ulla; Gyurcsányi, Róbert E.; Zebger, Ingo; Scheller, Frieder W.; Yarman, AysuThe epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.Öğe Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARSCoV-2 RBD protein(2022) Yarman, Aysu; Bognar, Zso´fia; Supala, Eszter; Zhang, Xiaorong; Bier, Frank F.; Scheller, Frieder W.; Gyurcsanyi, Robert E.We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (KD) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.Öğe Specific features of epitope-MIPs and whole-protein MIPs as illustrated for AFP and RBD of SARS-CoV-2(2024) Yarman, Ayşe; Zhang, Xiaorong; Kovács, Norbert; Bognár, Zsófia; Gyurcsányi, Róbert E.; Bier, Frank F.; Scheller, Frieder W.Molecularly imprinted polymer (MIP) nanofilms for alpha-fetoprotein (AFP) and the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 using either a peptide (epitope-MIP) or the whole protein (protein-MIP) as the template were prepared by electropolymerization of scopoletin. Conducting atomic force microscopy revealed after template removal and electrochemical deposition of gold a larger surface density of imprinted cavities for the epitope-imprinted polymers than when using the whole protein as template. However, comparable affinities towards the respective target protein (AFP and RBD) were obtained for both types of MIPs as expressed by the KD values in the lower nanomolar range. On the other hand, while the cross reactivity of both protein-MIPs towards human serum albumin (HSA) amounts to around 50% in the saturation region, the nonspecific binding to the respective epitope-MIPs is as low as that for the non-imprinted polymer (NIP). This effect might be caused by the different sizes of the imprinted cavities. Thus, in addition to the lower costs the reduced nonspecific binding is an advantage of epitope-imprinted polymers for the recognition of proteins.
