Protein-selective adsorbers by molecular imprinting via a novel two-step surface grafting method

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Molecularly imprinted polymers (MIP) offer in principle a robust, cost-efficient alternative to antibodies, but it is still a challenge to develop such materials for protein recognition. Here we report the molecular imprinting of a functional polymeric hydrogel layer with whole protein lysozyme as template in two-step grafting procedure by a novel initiation approach on track-etched (TE) polyethylene terephthalate (PET) and cellulose membrane surfaces. This two-step grafting strategy is based on surface functionalization with aliphatic C-Br groups which can be used as initiator for surface-initiated atom transfer radical polymerization (SI-ATRP) and photo-initiated copolymerization. At first, the scaffold poly(methacrylic acid) (PMAA) was obtained through SI-ATRP of poly(tert.-butyl methacrylate) (PtBMA) and subsequent hydrolysis. Thereafter, it was assembled with the template to form a stable PMAA/protein complex. In the second step, a polyacrylamide (PAAm) hydrogel was synthesized via surface UV-initiated grafting/crosslinking copolymerization around the scaffold/protein complex. Finally, the template was eluted to yield the grafted hydrogel layer with binding sites having complementary size, shape and appropriate arrangement of the functional groups to rebind the target protein. The selectivity of lysozyme recognition, relative to cytochrome C with similar size and isoelectric point, was increased by optimizations of scaffold chain length, UV grafting/crosslinking time and chemical crosslinking degree of the PAAm-based hydrogel on TE PET membrane. The feasibility for the development of protein MIP in a straightforward way by independent optimization of crucial parameters (structures of scaffold with functional groups and of crosslinked hydrogel matrix) has been demonstrated. The novel strategy of two-step grafting was further tested to develop immunoglobulin G imprinting on PET membrane. Immunoglobulin G has much bigger size than that of lysozyme. Through optimizations of scaffold and crosslinking degree, a satisfactory property of immunoglobulin G imprinted membrane was obtained and evaluated via comparison of binding properties for immunoglobulin G and human serum albumin. Mixture of immunoglobulin G and human serum albumin is an important biological background for protein separation and production. Thereafter, cellulose membrane with much more specific surface area and higher porosity than PET membrane was chosen as the new substrate for lysozyme imprinting. The imprinted structure was successfully synthesized on cellulose membrane via improvement of the UV-irradiation intensity and monomer concentration by UV-grafting/crosslinking. The property of the lysozyme imprinted cellulose membrane was improved via the similar optimization what has done during imprinting lysozyme on PET membrane. In order to improve the control of imprinted polymer structure, living polymerization method reversible addition fragmentation chain transfer polymerization (RAFT) was introduced to combine with ATRP instead of the conventional UV-irradiation of C-Br groups. ATRP initiator benzyl chloride and UV-RAFT initiator dithiocarbamate were immobilized on PET membrane surface and successfully characterized via their reactivity in surface-initiated ATRP and RAFT, respectively. The independency of the initiation of these two initiators was achieved on the surface covered with 50% benzyl chloride and 50% dithiocarbamate, this showed a good potential to be applied for the protein imprinting.
Lesezeichen:
Dokumententyp:
Wissenschaftliche Abschlussarbeiten » Dissertation
Fakultät / Institut:
Fakultät für Chemie » Technische Chemie
Dewey Dezimal-Klassifikation:
500 Naturwissenschaften und Mathematik » 540 Chemie
Beitragende:
Prof. Dr. Ulbricht, Mathias [Betreuer(in), Doktorvater]
Gutmann, Jochen Stefan [Gutachter(in), Rezensent(in)]
Sprache:
Englisch
Kollektion / Status:
Dissertationen / Dokument veröffentlicht
Promotionsantrag am:
12.07.2013
Dokument erstellt am:
17.10.2013
Dateien geändert am:
29.10.2013
Datum der Promotion:
16.10.2013
Medientyp:
Text