AVS2010 Session BI2-WeM: Proteins & Peptides on Surfaces

Utilization of biological molecules such as proteins in biosensing and biofuel cell applications is one of the most innovative research fields. A major challenge that needs to be overcome in these applications is the adequate stabilization of proteins. Porous silica material made by the sol-gel process provides a promising host matrix for the encapsulation of proteins to increase their stability. The main objective of this study is to elucidate protein-host matrix interactions for the stabilization of proteins. In our study, lysozyme and cytochrome c were used as model proteins. We have developed two novel vapor exposure sol-gel techniques to encapsulate proteins using tetra methyl orthosilicate (TMOS) as a precursor: (I) in-situ protein exposure method, where a buffer containing a protein is directly exposed to TMSO vapor, and (II) buffer exposure method, where a buffer was first exposed to TMOS vapor and then a protein was added. Additionally, organically-modified glasses are used to study the effect of host-matrix hydrophobicity on protein structure and stability. Circular dichroism and high resolution derivative UV spectroscopy are used to evaluate the structure and thermal stability of encapsulated proteins. The effect of protein concentration and sucrose (a model osmolyte) on the structure and stability of encapsulated proteins are characterized. Cytochrome c retained native-like structures while lysozyme becomes partly unfolded when encapsulated in the silica matrix. Entrapment generally increases the thermal stability of proteins. Proteins encapsulated via the in situ technique are found to have higher thermal stability compared to those encapsulated using method II. In general, method II gave rise to proteins with more secondary structure. However, proteins in this matrix are less resistant to thermal denaturation. In addition, there are concentration-dependent decreases in protein secondary structure when encapsulated by method II. We believe that the trends are due to protein adsorption onto silica which causes denaturation. Osmolytes shift the protein native state ensemble towards more compact conformations, thereby increasing the conformational stability of proteins. We observed enhanced secondary structure of cytochrome c with the addition of 0.5 M sucrose and this enhanced structure and stability is preserved when the protein is encapsulated in silica gel in the presence of sucrose. The effect of increasing the host-matrix hydrophobicity by incorporating alkyl group substituted alkoxysilanes into the silica matrix on protein structure and stability will be presented.

Commercial DNA array slides are commonly made by microprinting techniques. These nanoliter doplets evaporate within seconds and this fast drying may contribute to heterogeneous spots and inconsistent results. Successful development and optimization of DNA-functionalized surfaces for microarray and biosensor applications requires a better characterization of immobilized DNA. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) is applied to the study of spotted DNA surfaces on commercial microarray slides. ToF- SIMS results are directly compared to fluorescence images. Maximum Autocorrelation Factor (MAF) image analysis, a technique independent of the scaling of the raw data, is used to analyze the ToF-SIMS images. An IONTOF TOF.SIMS 5-100 instrument using a Bi source is used to analyze the samples. Immobilized DNA probes with 10, 20 and 40μM DNA concentrations as well as different Cy3 label concentrations are spotted on a commercially available microarray polymer slide. The effect of the spotting solution concentration as well as the amount of Cy3 label on spot uniformity is studied. MAF analysis of the ToF-SIMS image for a 20μM DNA spot shows that the areas seen as having high fluorescence intensities are related to higher concentrations of phosphate groups (from the DNA backbone) as well as sulfates and peaks with masses corresponding to the DNA bases. A comparison of MAF analysis of ToF-SIMS images for different DNA spotting concentrations indicates that the concentration of the spotting solution has an effect on the uniformity of the spot.

Synthetic materials are often used for biomedical applications. Interaction of cells with the interfaces and tissue components determine the biological outcome of the device. Knowledge about the interaction between the cells and biointerfaces is hence of importance in area such as biomaterials, tissue engineering and cell culture. The interaction of the cells with its surroundings is mediated at the molecular and macromolecular level. Specific interaction with the extracellular matrix components or macromolecules in the outer membrane of adjacent cells provides signaling and communication pathways. Here patterns of extracellular matrix protein are used to study the development of cellular adhesion complexes.

Protein nanopatterns at the 100-3000 nm scale and with lateral ordering between independent ligands and controlled lateral mobility has been made by using a nanoscale chemical contrast of Au patches in a background of SiO₂ by colloidal lithography. The nanostructured surfaces are made by depositing a triple polyelectrolyte layer (PDDA/PSS/PAX) at Au substrates. Latex particles self assemble at the surface governed by electrostatic forces followed by SiO₂ evaporation and removal of the particles. The generated short range ordered arrays were further modified by octadecylmercaptane adsorption. The samples were subsequently treated with PLL-g-PEG for 30 min. followed by adsorption of fibronectin for 2 h. Myoblast cell(C2C12) or MDA-MB-435 cells were added to the samples and allowed to adhere to the surfaces for 6 h or 24 hours.

Fibronectin distribution at the nanopatterned surfaces was studied via liquid AFM showing that protein were adsorbed preferentially on the alkane thiol patches. SEM images showed that protein was patterned over large areas. Protein patterns of several other proteins such as Osteopontin, Vitronectin and Laminin were also demonstrated. Fluorescent microscopy showed that cells adhered to the patterns of size from 200nm and up. Small focal complexes were observed at the 200nm structures which were not linked to the actin cytoskeleton. For 500nm and 1000nm patchs cell showed small focal adhesions connected to thin actin fibers and the adhesions were limited to individual patches.