AVS2009 Session BI+AS+NS-ThA: Micro and Nanoengineering of Biointerfaces I

Signal transduction in living cells is carried out through cascades of chemical reactions, which generally begin on the cell membrane surface.

In recent years, there has been growing realization that the large-scale spatial arrangement of cell surface receptors can regulate the outcome of ensuing signal transduction process. Signaling through the T cell receptor (TCR) in the context of the immunological synapse provides a case in point. Spatial reorganization of TCRs occurs on multiple length-scales, and apparently with multiple purposes, during antigen recognition by T cells. The cell membrane and cytoskeleton, working as an inseparable unit in this case, create the mechanical framework within which TCR signaling processes occur. To better study these phenomena, a new experimental strategy, in which the spatial positions of cell membrane receptors are directly manipulated through mechanical means, has emerged. By physically inducing a 'spatial mutation' of the signali ng apparatus, the role of spatial organization in signal transduction as well as the mechanisms by which it arises can be illuminated. Specific applications of this strategy to TCR signaling and other cell-cell signaling systems will be discussed.

Supported lipid bilayers (SLB) constitute a simple model of cell membranes and are of particular interest as components of future generations of biosensors based on transmembrane proteins. Techniques which are able to produce arrays with small micrometer-sized sensor areas in a cheap and fast way are beneficial. A major challenge producing such arrays of SLBs is their need for an aqueous environment during formation and operation, which has so far prevented the wide-spread use of common techniques to produce arrays such as spotting.

Poly(ethylene glycol) (PEG) can be incorporated into the membrane of liposomes through lipid molecules end-functionalized with a PEG chain and these liposomes were shown to spontaneously fuse to PEG-SLB on glass surfaces with a highly hydrated PEG cushion on each side of the membrane allowing ample space and protection for incorporation of membrane proteins[1,2]. Since Cremer and coworkers [3] also showed that with increasing PEG concentration the air-stability of PEG-SLBs increases it is a very promising SLB system to use for spotting where membrane air exposure during processing is a severe constraint.

We present an investigation of the limits to spontaneous PEG-SLB in terms of PEG-lipid density, demonstrating that crossing the mushroom-to-brush regime of polymer concentration prevents the PEG-SLB formation due to steric effects and shielding of the interactions². Furthermore, we present conditions under which formation of PEG-SLBs is facilitated and can proceed by liposome fusion also in the brush regime as well as characterization of the kinetics of formation and the structure of these PEG-SLBs. The use of such buffers and liposomes for production of membrane arrays on glass using a non-contact piezo-spotter was then explored in detail in order to find optimal conditions of buffer composition and PEG concentration.

Technologies utilizing arrays of immobilized biomolecules on planar surfaces are emerging as powerful high throughput tools for bioanalytical measurements. Nowadays, optical sensors based on fluorescence detection are the most widespread. However, many applications especially in the area of protein sensing, rely on the availability of optimized sensing interfaces and signal amplification strategies. Liposomes, due to their hollow particle-like structure and their unique chemical and physical properties, have greatly contributed to the development of sensitive and accurate biological assays.

In this presentation, we highlight with results obtained recently, the contributions of phospholipid vesicles to the development of high performance fluorescence based biosensors.

First, liposomes are an optimal platform for the surface immobilization of membrane proteins since they provide the natural environment required for the functional surface immobilization of these fragile molecules. We demonstrate the creation of a functional, heterogeneous array of G-protein coupled receptors. Vesicles obtained from cellular membrane extracts containing either the H1R-histamine receptor or the M2R-muscarinic receptors were immobilized on a conventional oligonucleotide microarray via complementary tags. Fluorescent ligand binding assays were then performed illustrating that the receptors kept their native conformation. As an alternative platform, we introduce a novel approach for the creation of vesicle multilayers using zirconium phosphate chemistry. As demonstrated in a model biomolecular binding assay, such three dimensional constructs increase the protein loading capacity of a sensor surface.

Liposomes are also excellent candidates as labels for biological assays: phosphocholine-based vesicles are non-fouling and biomolecules or marker molecules (e.g. fluorophores or enzymes) can be easily attached to their surface or encapsulated in their inner cavity. We show that fluorescently labeled phospholipid vesicles provide simple and cheap means for signal amplification and sensitive protein detection on a microarray format. Using vesicles, up to 100 fold increase in sensitivity was observed in a model protein microarray with confocal read-out, compared to a conventional assay performed with fluorophore labeled antibodies.

The various approaches presented here will contribute to the development of sensitive and high performance microarrays for a variety of applications including the investigation of membrane proteins.

Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has been shown to be very sensitive for surface analysis and has been very useful for trace element detection. With the advent of improved analyzers, imaging ToF-SIMS provides spatial distribution of different species and helps in plotting the surface reactivity maps. Use of multivariate analysis, especially Principal Component Analysis (PCA) makes this technique even more powerful by differentiating regions with different chemistries. ToF-SIMS and PCA has been used in this work to study a very important two component patterned chemistry, which can have applications in bio-chips and cell-based biosensors. The chemistry is based on n-hydroxysuccinimide (NHS) esters; these molecules are widely used as leaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces. The present work utilizes our knowledge of a previously studied model system, where NHS molecules were self-assembled on a gold substrate, and XPS and ToF-SIMS was used to characterize and understand the effect of hydrolysis and regeneration. We have demonstrated the extension of this chemistry to a commercial poly(ethylene glycol) (PEG)-based polymer films coated on glass slides. NHS and methoxy-capped regions were co-patterned onto these slides using photolithographic methods; then imaged with ToF-SIMS/PCA. NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the matrix chemistry. Surface-specific protein coupling was observed by surface-selective reaction of streptavidin with the NHS patterns. The next step involved the preparation of photolithographic patterns of two affinity ligands (biotin and chloroalkane) for the specific immobilization of two different proteins (Streptavidin and HaloTag®). Spontaneous formation of high-fidelity surface patterns of the two proteins from their mixed solution was observed and characterized. In addition to Streptavidin and HaloTag®, ToF-SIMS detected the presence of non-specific BSA adsorption, a masking protein present in excess in the protein solutions, onto the patterned surfaces. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. However PCA of the ToF-SIMS data, improves discrimination of ions specific to each protein, facilitating surface pattern discrimination based on protein type. Also, ToF-SIMS imaging detected regions where residue from incompletely removed UV-exposed photoresist was present and its influence on protein adsorption.