Fabrication of Robust (Bio)interfaces Based on Reactive Polymer Films: Surface Confinement, Reactivity and Pattern Fabrication on Multiple Length Scales
The aim of the work described in this Thesis was to investigate interfacial reactions in confinement on ultrathin homopolymer and diblock copolymer films, the immobilization of (bio)molecules and the fabrication of biomolecular patterns by reactive microcontact printing (mCP) on these reactive polymer films. Taking advantage of the microphase separation of diblock copolymer films, the fabrication of nanopatterns was investigated, which could contribute to the future development of a model system that enables one to area-selectively deposit (write) and address (read out) (bio)molecules. Chapter 2 presented an overview of (bio)reactive surfaces and biointerfaces based on organic and polymeric films, their characterization, as well as surface reactions and patterning of these films. The mCP technique was introduced in detail owing to its central role in this Thesis as a flexible approach for the production of micrometer and sub-micrometer scale patterns. Block copolymers were also discussed as materials used in a “bottom-up” approach to prepare nanometer scale patterns by exploiting the characteristic microphase separation. In Chapter 3, the effect of the spatial confinement of the reactants on the kinetics of the hydrolysis of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) and polystyrene-block-poly(tert-butyl acrylate) (PSn-b-PtBAm) ultrathin films was systematically investigated. The activation energies determined according to the Arrhenius equation, and in particular the activation entropies calculated according to the transition state theory, revealed that steric crowding in the surface-near region and tightness of the transition state is less pronounced in the polymer films compared to related self-assembled monolayers (SAMs) that expose the same reactive ester groups. Apparent rate constants calculated according to Fourier transform infrared (FTIR) spectroscopy and contact angle (CA) data for both polymer films and SAMs directly demonstrated that the polymer films are characterized by higher reactivity, as well as a higher density of reactive functional groups near and at the polymer surface. However, the reactivity on polymer films was reduced compared to reactivity in solution because of restricted access of reactants and reduced mobility of the ester functional groups in these films. Finally, it was found that polymer film thickness, thermal pre-treatment of the films, block copolymer composition for PSn-b-PtBAm and local surface composition did not affect the rate constants. Spin-coated thin films of PNHSMA were investigated in Chapter 4 as reactive layers for obtaining platforms for biomolecule immobilization with high molecular loading. The surface reactivity of PNHSMA films in coupling reactions with amino-functionalized poly(ethylene glycol) (Mn: 500 g/mol) (PEG500-NH2) was determined by FTIR spectroscopy, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy and ellipsometry measurements, respectively. The PEG500-NH2 loading observed was about three times higher for the polymer thin films compared to SAMs of 11,11´-dithiobis(N-hydroxysuccinimidyl undecanoate) (NHS-C10) on Au. These data indicate that the coupling reactions are not limited to the outermost surface layer of the polymer films, but proceed into the surface-near regions of the films. An increased loading was also observed by surface plasmon resonance (SPR) measurements for the covalent immobilization of amino-functionalized probe DNA. Hybridization of fluorescently labeled target DNA was successfully detected by fluorescence microscopy and surface plasmon resonance-enhanced fluorescence spectroscopy (SPFS), thereby demonstrating that thin films of PNHSMA show robustness and comprise an attractive and simple platform for the immobilization of biomolecules with high molecular densities. Finally, the successful application of PNHSMA films as platform for biosensors for pathogen detection was demonstrated using a protein G mediated antibody-based detection of listeria. The investigation of PS690-b-PtBA1210 films and their derivatization to obtain tailored biointerfaces was presented in Chapter 5. Derivatized PS690-b-PtBA1210 films showed good stability under a broad range of conditions. Hydrolysis of the reactive t-butyl ester groups was performed in trifluoroacetic acid, 3M aqueous HCl, and in the gas phase (HCl). After the subsequent activation with NHS ester groups a variety of amino-functionalized (bio)molecules were covalently immobilized on the previously hydrolyzed surfaces. The reactivity of the PS690-b-PtBA1210 films and in particular the controllable loading with amino functionalized PEG500-NH2 were studied by FTIR and XPS. Subsequently, the immobilization of biologically relevant molecules, such as bovine serum albumin (BSA) and poly(L)lysine (PLL), on PS690-b-PtBA1210 films were studied by fluorescence microscopy. To demonstrate possible applications of the PS690-b-PtBA1210 based platform as viable biointerfaces, hybridization of target DNA with previously covalently immobilized probe DNA, as well as the interaction of two different types of cells, i.e. K562 and pancreatic cancer cells, on functionalized PS690-b-PtBA1210 films were investigated. In Chapter 6, the fabrication of robust biomolecule microarrays on spin-coated thin films of PNHSMA on oxidized silicon and glass by reactive mCP was described. The approach combines the advantages of activated polymer thin films as coupling layers, characterized by high reactivity and high molecular loading (Chapters 3 and 4), with the versatility and flexibility of soft lithography. The transfer of amino end functionalized PEG500-NH2 from oxidized poly(dimethyl siloxane) (PDMS) elastomer stamps to PNHSMA films was shown by FTIR spectroscopy, XPS, fluorescence microscopy and ellipsometry measurements to result in covalent coupling and identical grafting densities as reported in Chapter 4 for coupling from solution. The PEG-protected areas effectively inhibited the adsorption of fluoresceinamine, BSA, as well as 25-mer DNA, while the unreacted NHS ester groups retained their reactivity towards primary amino groups. Biomolecule microarrays were thus conveniently fabricated in a 2-step procedure. The hybridization of target DNA to immobilized probe DNA in micropatterns proved the concept of reactive mCP on activated polymer films for obtaining robust patterned platforms for biomolecule immobilization and screening. Three different lithographic approaches to produce chemical patterns on ultrathin PS690-b-PtBA1210 films were introduced in Chapter 7, which were expanded to obtain patterns of biomolecules with (sub)micrometer feature sizes. In approach (A), PS690-b-PtBA1210 films were homogeneously hydrolyzed and subsequently activated with NHS. Fluoresceinamine and BSA were patterned and covalently bound on the activated polymer films in sequential direct molecular transfer steps using reactive μCP. NHS functionalized polymer films were also patterned with PEG500-NH2 by reactive μCP in approach (B). The PEG layer was used as antifouling layer to prevent the non-specific adsorption of (bio)molecules in the subsequent covalent coupling step of fluoresceinamine and BSA carried out in solution. The area selective immobilization was also successfully demonstrated for 25-mer probe DNA, as shown by the fluorescence microscopic detection of the hybridization of dye-labeled target DNA. In approach (C), the polymer films were firstly locally hydrolyzed with trifluoroacetic acid that was locally applied on the films using acid soaked PDMS stamps. A detailed study of the reactive μCP mechanism led to the conclusion that ink spreading and diffusion must be controlled for faithful pattern transfer, in particular on the sub-mm level. In addition, it was found that patterns with micrometer scale dimensions could be fabricated by using stamps with > 10 mm dimensions by controlling the spreading of trifluoroacetic acid. Thus, ultrahigh density patterns could be conveniently fabricated. In Chapter 8, nanofabrication and the subsequently selective immobilization of (bio)molecules on reactive PS690-b-PtBA1210 ultrathin films were studied. As revealed by AFM and spectroscopic techniques, the surface exposed PtBA islands in a matrix of unreactive PS. The films were then hydrolyzed with trifluoroacetic acid and activated with NHS. The domain selective immobilization of fluoresceinamine on the films was globally analyzed by fluorescence microscopy and was also investigated on the nanometer scale by AFM adhesion force mapping in the force-volume mode. Finally, the area selective functionalization of BSA and polyamidoamine (PAMAM) dendrimers on nanopatterned block copolymer thin films on the micrometer and sub-micrometer scales was carried out by reactive microcontact printing and the dip-pen nanolithography technique, respectively, to yield surfaces that are patterned with (bio)molecules on multiple length scales. Therefore the polymer thin film platforms and patterning approaches investigated herein provide the opportunity to study a broad variety of surface-mediated biological recognition processes in the future.