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AuthorGast, Manueladc.contributor.author
Date of accession2020-10-20T14:18:02Zdc.date.accessioned
Available in OPARU since2020-10-20T14:18:02Zdc.date.available
Year of creation2019dc.date.created
Date of first publication2020-10-20dc.date.issued
AbstractRapid detection of viral contaminations remains a challenging task in various fields including viral clearance studies for biopharmaceuticals, clinical diagnostics, detection of biological agents or checking of food-borne pathogens. The majority of the currently available virus detection assays such as cell cultures or enzyme-linked immunosorbent assays (ELISA) are still laborious and energy-consuming strategies combined with excessive costs. Hereby, the latter is based on the ability of antibodies to bind specifically at the surface of a target virus, the antigen, with high affinity and selectivity. Inspired by the ‘lock-and-key’ principle in nature, this recognition process was translated into synthetic systems generating materials with similar molecular recognition properties. Ever since the first reports on such synthetically designed systems with ‘molecular memory’ called molecularly imprinted polymers (MIPs) or materials, this field has rapidly expanded. Nowadays, templates are recruited from small molecules to macromolecules and even biological targets, i.e. proteins, viruses, cells. In general, molecular imprinting can be described as creation of synthetic polymers with biomimetic molecular recognition properties for a selected template. Hereby, the template directs the assembly and orientation of monomer(s) via a crosslinking agent. Following polymerization and template extraction, binding sites with complementary size, shape, and functional group arrangement in relation to the template molecule are obtained. Although the design of synthetic affinity matrices with recognition properties for biological targets, i.e. viruses, still remains challenging, various successful attempts have been reported. The major obstacles when imprinting virus targets are the fragility, dimension, and limited solubility of the viral target, as well as the restricted availability of pure virus material and the need of appropriate laboratories and personnel. In turn, molecularly imprinted polymers for viruses convince with their high sensitivity and affinity, ease of production, saving of time and costs, robustness of the material, and long-term stability. Hence, molecularly imprinted polymers with ‘virus memory’ represent an ideal tool for the detection, enrichment, or removal of such pathogens. Most successful virus imprinted polymers rely on surface imprinting approaches. These entail specific binding sites predominantly present at the surface of the material. Hence, mass transfer limitations are minimized, and rapid diffusion/kinetics of the template are ensured. In the present thesis, the development and optimization of an adjustable, synthetic surface molecular imprinting strategy yielding virus-selective core-shell particles have been pursued. Human Adenovirus, which belongs to the category of non-enveloped viruses with icosahedral morphology was used as model virus within the present thesis. The developed imprinting approach is composed of sequential steps initiated by the template immobilization at the glutaraldehyde-functionalized carrier material, i.e. sub-micrometer particles. Next, a polymer shell was grown from the surface of the particles embedding the virus templates within this layer. Finally, the virus was extracted creating virus-selective binding sites at the surface of the carrier matrix. The research described within the present thesis contributes to innovative imprinting strategies for viral species addressing the previously mentioned challenges. The obtained virus imprinted polymers (VIPs) were characterized for their ability to specifically recognize the initial template virus by comparison with non-imprinted (i.e. control) polymers (NIPs). Single and duplex batch rebinding assays distinctly revealed high affinity and selectivity of those materials. Minute Virus of Mice (MVM) was used for competitive studies and virus quantification was performed via quantitative polymerase chain reaction (qPCR). Furthermore, super-resolution fluorescence microscopy studies confirmed the presence of virus particles at the imprinted bead surface and scanning electron microscopy images provided morphological insight on these matrices. Biological risks during the synthesis procedure were minimized in a next development step by using a multi-subunit protein complex as template mimicking the native virus structure instead of pathogenic virus particles. Although the imprinted binding sites were indeed established using hexon protein – a surface capsid protein of Adenoviruses – the resulting polymer matrix was able to bind the relevant infectious virus with excellent affinity and selectivity. Furthermore, highly purified human Adenovirus type 5 was prepared by a newly developed, fast, and robust chromatographic purification strategy using novel virus purification materials. This purified virus material was characterized in detail and was studied for the presence of virus aggregates. For that purpose, quantitative polymerase chain reaction (qPCR), gel electrophoresis (SDS-PAGE), and nanoparticle tracking analysis (NTA) were used. Adenovirus obtained via this procedure was then used for imprinting and fluorescence imaging purposes. The presented strategies are fundamental steps towards the creation of synthetic receptor materials for selective virus capture establishing molecular imprinting of biological templates as a versatile and generic technology. Hence, it may be adapted to a wide variety of biological species including enveloped viruses.dc.description.abstract
Languageendc.language.iso
PublisherUniversität Ulmdc.publisher
Articles in publ.Gast et al., Advances in Imprinting Strategies for Selective Virus Recognition – A Review, Trends in Analytical Chemistry, 114, (2019) 218-232; DOI: 10.1016/j.trac.2019.03.010dc.relation.haspart
Articles in publ.Gast et al., Understanding the viral load during the synthesis and after rebinding of virus imprinted particles via real-time quantitative PCR, Analyst, 143 (11), (2018) 2616-2622; DOI: 10.1039/c8an00300adc.relation.haspart
Articles in publ.Gast et al, Enhanced Selectivity by Passivation: Molecular Imprints for Viruses with Exceptional Binding Properties, Analytical Chemistry, 90 (9), (2018) 5576−5585; DOI: 10.1021/acs.analchem.7b05148dc.relation.haspart
Articles in publ.Gast et al., Selective virus capture via hexon imprinting, Martials Science and Engineering C, 99, (2019) 1099-1104; DOI: 10.1016/j.msec.2019.02.037dc.relation.haspart
Articles in publ.Gast et al., Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core-Shell Matrixes, Analytical Chemistry, 92, (2020) 3050−3057; DOI: 10.1021/acs.analchem.9b04328dc.relation.haspart
Articles in publ.Gast et al., Nanoparticle Tracking of Adenovirus by Light Scattering and Fluorescence Detection, Human Gene Therapy Methods, 30 (2019), 245-244; DOI: 10.1089/hgtb.2019.172dc.relation.haspart
LicenseStandard (ohne Print-on-Demand)dc.rights
Link to license texthttps://oparu.uni-ulm.de/xmlui/license_opod_v1dc.rights.uri
KeywordVirusdc.subject
KeywordQuantitative Polymerase Chain Reactiondc.subject
KeywordEpitop Imprintingdc.subject
KeywordHexon Proteindc.subject
KeywordAdenovirusdc.subject
KeywordStimulated Emission Depletion (STED) Microscopydc.subject
KeywordVirus Purificationdc.subject
KeywordNanoparticle Tracking Analysis (NTA)dc.subject
Dewey Decimal GroupDDC 610 / Medicine & healthdc.subject.ddc
LCSHMolecular imprintingdc.subject.lcsh
LCSHAntigenic determinantsdc.subject.lcsh
LCSHAdenovirusesdc.subject.lcsh
LCSHNanoparticlesdc.subject.lcsh
MeSHVirusesdc.subject.mesh
TitleAdvanced molecular imprinting strategies for virusesdc.title
Resource typeDissertationdc.type
Date of acceptance2020-07-30dcterms.dateAccepted
RefereeMizaikoff, Borisdc.contributor.referee
RefereeWalther, Pauldc.contributor.referee
DOIhttp://dx.doi.org/10.18725/OPARU-33415dc.identifier.doi
PPN1736065327dc.identifier.ppn
URNhttp://nbn-resolving.de/urn:nbn:de:bsz:289-oparu-33477-4dc.identifier.urn
GNDVirendc.subject.gnd
GNDPolymerase-Kettenreaktiondc.subject.gnd
GNDEmissionsverringerungdc.subject.gnd
FacultyFakultät für Naturwissenschaftenuulm.affiliationGeneral
InstitutionInstitut für Analytische und Bioanalytische Chemieuulm.affiliationSpecific
InstitutionZE Elektronenmikroskopieuulm.affiliationSpecific
Grantor of degreeFakultät für Naturwissenschaftenuulm.thesisGrantor
DCMI TypeTextuulm.typeDCMI
CategoryPublikationenuulm.category
University Bibliographyjauulm.unibibliographie


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