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This study introduces a thermally responsive, polymer-based electrode system. The key component is a surface-attached, temperature-responsive poly(oligoethylene glycol) methacrylate (poly(OEGMA)) type polymer bearing photoreactive benzophenone and carboxy groups containing side chains. The responsive behavior of the polymer in aqueous media has been investigated by turbidimetry measurements. Polymer films are formed on gold substrates by means of the photoreactive 2(dicyclohexylphosphino)benzophenone (DPBP) through photocrosslinking. The electrochemical behavior of the resulting polymer-substrate interface has been investigated in buffered [Fe(CN)6](3-)/[Fe (CN)6](4-)solutions at room temperature and under temperature variation by cyclic voltammetry (CV). The CV experiments show that with increasing temperature structural changes of the polymer layer occur, which alter the output of the electrochemical measurement. Repeated heating/cooling cycles analyzed by CV measurements and pH changes analyzed by quartz crystal microbalance with dissipation monitoring (QCM-D) reveal the reversible nature of the restructuring process. The immobilized films are further modified by covalent coupling of two small biomolecules - a hydrophobic peptide and a more hydrophilic one. These attached components influence the hydrophobicity of the layer in a different way the resulting change of the temperature-caused behavior has been studied by CV indicating a different state of the polymer after coupling of the hydrophobic peptide.
Para-maleimidophenyl (p-MP) modified gold surfaces have been prepared by one-step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N-terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p-MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read-out for a broad variety of biomolecular interactions on the same chip. Copyright (c) 2014 John Wiley & Sons, Ltd.
Das Influenzavirus infiziert Säugetiere und Vögel. Der erste Schritt im Infektionszyklus ist die Anbindung des Viruses über sein Oberflächenprotein Hämagglutinin (HA) an Zuckerstrukturen auf Epithelzellen des respiratorischen Traktes im Wirtsorganismus. Aus den drei komplementaritätsbestimmenden Regionen (complementarity determining regions, CDRs) der schweren Kette eines monoklonalen Hämagglutinin-bindenden Antikörpers wurden drei lineare Peptide abgeleitet. Die Bindungseigenschaften der drei Peptide wurden experimentell mittels Oberflächenplasmonenresonanzspektroskopie untersucht. Es zeigte sich, dass in Übereinstimmung mit begleitenden Molekulardynamik-Simulationen zwei der drei Peptide (PeB und PeC) analog zur Bindefähigkeit des Antikörpers in der Lage sind, Influenzaviren vom Stamm X31 (H3N2 A/Aichi/2/1968) zu binden. Die Interaktion des Peptids PeB, welches potentiell mit der konservierten Rezeptorbindestelle im HA interagiert, wurde anschließend näher charakterisiert. Die Detektion der Influenzaviren war unter geeigneten Immobilisationsbedingungen im diagnostisch relevanten Bereich möglich. Die Spezifität der PeB-Virus-Bindung wurde mittels geeigneter Kontrollen auf der Seite des Analyten und des Liganden nachgewiesen. Des Weiteren war das Peptid PeB in der Lage die Bindung von X31-Viren an Mimetika seines natürlichen Rezeptors zu inhibieren, was die spezifische Interaktion mit der Rezeptorbindungsstelle im Hämagglutinin belegt. Anschließend wurde die Primärsequenz von PeB durch eine vollständige Substitutionsanalyse im Microarray-Format hinsichtlich der Struktur-Aktivitäts-Beziehungen charakterisiert. Dies führte außerdem zu verbesserten Peptidvarianten mit erhöhter Affinität und breiterer Spezifität gegen aktuelle Influenzastämme verschiedener Serotypen (z.B. H1N1/2009, H5N1/2004, H7N1/2013). Schließlich konnte durch Verwendung einer in der Primärsequenz angepassten höher affinen Peptidvariante die Influenzainfektion in vitro inhibiert werden. Damit stellen die vom ursprünglichen Peptid PeB abgeleiteten Varianten Rezeptormoleküle in biosensorischen Testsystemen sowie potentielle Wirkstoffe dar.
Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.
Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.