RESUMO
A novel method to produce sub-microwalled chemically activated polymer microwells by one-step UV-lithography under ambient conditions which are selectively coated with gelatin is introduced. The dimensions as well as the shape of the resulting polystyrene structures are both tunable merely by the irradiation time through one and the same mask. It is shown that the UV-irradiation initiates three effects at those surface areas which are not covered by the mask: (i) oxidation, (ii) cross-linking, and (iii) degradation of polystyrene. The superposition of those effects results in the formation of microscaled, oxidized polymer wells separated by polymer walls, whereas the polymer walls are formed below the mask structures. Topographical changes induced by the UV-irradiation are investigated by atomic force microscopy after different irradiation times. It is shown by X-ray photoelectron spectroscopy and ellipsometric investigations that the chemical composition of the irradiated areas and the degradation of polystyrene reach an equilibrium state after an irradiation time of 10 min. The lateral distribution of the cross-linked and oxidized and of the nonmodified polystyrene after irradiation was determined by fluorescence microscopy and time-of-flight secondary ion mass spectrometry. After the irradiated samples were treated with gelatin solution, it was found that stem cells selectively attach to the irradiated areas. This is due to the selective immobilization of the gelatin on the irradiated polymer areas, which was proved by X-ray photoelectron spectroscopy experiments.
Assuntos
Desenho de Fármacos , Polímeros/química , Polímeros/metabolismo , Células-Tronco/citologia , Raios Ultravioleta , Animais , Adesão Celular , Técnicas de Cultura de Células , Camundongos , Microscopia de Fluorescência , Espectroscopia Fotoeletrônica , Propriedades de SuperfícieRESUMO
With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (microCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide - even for the preparation of peptide-functionalized small surface areas.
Assuntos
Adesão Celular , Isotiocianatos/química , Oligopeptídeos/química , Animais , Sequência de Bases , Proliferação de Células , Células Cultivadas , Primers do DNA , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A method for protein immobilization onto modified CdSe/ZnS quantum dot surfaces was developed using simple SNAP tag methodology.
Assuntos
Compostos de Cádmio/química , Nanopartículas/química , O(6)-Metilguanina-DNA Metiltransferase/química , Proteínas/química , Compostos de Selênio/química , Sulfetos/química , Compostos de Zinco/química , Caderinas/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/química , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Pontos Quânticos , Propriedades de SuperfícieRESUMO
Tobacco mosaic virus (TMV) is a tube-shaped, exceptionally stable plant virus, which is among the biomolecule complexes offering most promising perspectives for nanotechnology applications. Every viral nanotube self-assembles from a single RNA strand and numerous identical coat protein (CP) subunits. Here we demonstrate that biotechnologically engineered RNA species containing the TMV origin of assembly can be selectively attached to solid surfaces via one end and govern the bottom-up growth of surface-linked TMV-like nanotubes in situ on demand. SiO(2) wafers patterned by polymer blend lithography were modified in a chemically selective manner, which allowed positioning of in vitro produced RNA scaffolds into predefined patches on the 100-500 nm scale. The RNA operated as guiding strands for the self-assembly of spatially ordered nanotube 3D arrays on the micrometer scale. This novel approach may promote technically applicable production routes toward a controlled integration of multivalent biotemplates into miniaturized devices to functionalize poorly accessible components prior to use. Furthermore, the results mark a milestone in the experimental verification of viral nucleoprotein complex self-assembly mechanisms.
Assuntos
Nanotecnologia/métodos , Nanotubos/química , RNA/química , Vírus do Mosaico do Tabaco/metabolismo , Aldeídos/química , Biotecnologia/métodos , DNA/química , Microscopia de Força Atômica/métodos , Modelos Químicos , Conformação Molecular , Nucleoproteínas/química , Polímeros/química , Polimetil Metacrilato/química , Poliestirenos/química , RNA Ligase (ATP)/química , Silício/química , Dióxido de Silício/químicaRESUMO
Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) was applied to validate GRGDS peptide patterned surfaces. The structuring of the surfaces included several steps: micro contact printing (microCP), chemical etching and aminofunctionalization followed by chemical coupling of spacer-linked GRGDS peptides via an isothiocyanate anchor. TOF-SIMS analysis of characteristic ions and molecular fragments with a lateral resolution of 100 nm allowed proving the change in chemical properties of the surface with each step during the structuring process. We found that the application of polydimethylsiloxane as stamp material resulted in the contamination of the surface with this polymer. TOF-SIMS investigations, however, also showed that during the preparation process the contaminations were removed and do not influence the bio functionality of the surface patterns. The results of the surface analysis carried out with TOF-SIMS were confirmed by complementary cell adhesion experiments with murine fibroblasts. As a result, specific cell adhesion restricted to GRGDS peptide functionalized areas was obvious by the formation of focal adhesion contacts in the fibroblasts. Thus, TOF-SIMS is the method of choice in chemical characterization of surfaces in structuring and functionalization processes, because it offers the opportunity to follow surface contamination during the preparation process and to assess the influence of the contamination on the applicability of the final substrate.