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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 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.
