Increased Stability and Complexity of Glycol-Terminated Self-Assembled Monolayers and their Use in Supported Lipid Bilayers

Self-assembled monolayers (SAMs) terminated with a poly(ethylene glycol) moiety are able to resist non-specific protein and cell adhesion, which has made them an important class of surfaces for biochemical studies on solid substrates. Gold-thiolate SAMs have been used extensively for biochemical studies at a surface, since they are easy to form, easy to pattern, and gold is a biocompatible substrate. However, for patterned cell culture, typical glycol-terminated gold-thiolate SAMs are only stable for a maximum of a week, which does not allow for long-term biochemical processes to be studied. Increasing the intramolecular forces between the SAM monomers, by either hydrogen bonding or dipole-dipole interactions, has been demonstrated to increase pattern fidelity for up to five weeks in culture. Furthermore, it was found that gold topology plays a major role in the stability of these patterned cell-culture substrates. Additionally, increasing the control over the attachment of multiple proteins on a surface would allow for the generation of more complex protein maps. In order to control the relative concentration of two proteins on a surface, two pairs of hetero-functional glycol molecules were synthesized. Each pair contained a surface-reactive molecule and a protein-reactive molecule that could be coupled together by either a triazole ring or oxime bond. Varying the ratio of the surface reactive linkers then controls the ratio of two proteins binding to a surface, assuming that the two surface-reactive linkers have a similar affinity to couple to the surface. Functionalization of carboxylic acid-terminated SAMs with the two pairs of linkers was confirmed by reflective infrared spectroscopy. However, when these linkers were used to bind two different extracellular matrix proteins to the surface for cell attachment and growth, there was no observed attachment of cells. Glycol-terminated SAMs also provide an ideal cushion for the formation of a supported lipid bilayer (SLB), since they allow the bilayer to maintain its maximum fluidity due to the inertness of the glycol-terminated surface. Due to this inertness, typical methods for SLB formation do not work for SLB formation on a glycol-terminated SAM. Microcontact printing was used to pattern attachment sites for lipid vesicles to adhere and subsequently rupture to form SLBs on glycol-terminated SAMs. The formation and fluidity of these patterned SLBs were confirmed by a variety of surface-characterization techniques. The ability to form SLBs on a glycol-terminated SAM raises questions about how this occurs mechanisticly. Imaging surface plasmon resonance (SPRi) was used to monitor the rate of formation of the supported lipid monolayer (SLM) for the alkane-terminated regions of the monolayer and SLB formation for the glycol-terminated regions of the monolayer. The data supported a simultaneous attachment and rupture mechanism for SLM formation, while SLB formation occurs by attachment at the interface of the two SAM regions, followed by a slower spreading process over the glycol-terminated SAM. Ligand-gated ion channels directly couple small molecule binding to an electrical signal and making them a model system for biosensing, however instability of electrophysiology measuring has prevented their use as biosensors. SLBs provide a more stable lipid bilayer compared to traditional black lipid membranes, whose bilayer\u27s are formed over a small aperture, but creating a SLB device to measure ion-channel activity is not trivial. The strategy for SLB formation on patterned monolayers was utilized to create SLBs on glycol-terminated SAM gold microelectrodes that were surrounded by aluminum oxide which was coated with an alkane-terminated SAM. A double lift-off procedure was used to fabricate the microelectrode device, and SLB formation was monitored electrically. Furthermore, the pore-forming peptide alamethicin was inserted into the SLB and its activity was monitored using a voltage-clamp experiment

Mechanistic Insight into Patterned Supported Lipid Bilayer Self-Assembly

Patterned supported lipid bilayers (SLBs) provide a model system for studying fluid lipid bilayers and transmembrane proteins in an array format. SLB arrays self-assemble on patterned self-assembled monolayers (SAMs) consisting of hexadecanethiol and glycol-terminated regions. While the mechanism of SLB formation on glass has been studied extensively, the formation of SLBs on other substrates is not necessarily well understood. Moreover, SLB arrays on patterned SAMs represent an intriguing system, since lipid vesicles do not adhere to glycol-terminated monolayers. Here, we utilize surface plasmon resonance imaging (SPRi) and kinetic analysis to examine the mechanism of SLB formation on the glycol-terminated regions of patterned SAMs and supported lipid monolayer (SLM) formation on alkyl-terminated regions of patterned SAMs. We determine that vesicles rupture to form a patterned SLB through a two-step mechanism that is dependent upon vesicle attachment at the interface of the two regions of the patterned monolayer

Relative humidity sensors based on an environment-sensitive fluorophore in hydrogel films

A fluorescence-based sensing scheme exploiting an environment-sensitive fluorophore embedded in a hydrogel has been developed for measurement of relative humidity (RH). The fluorophore, dapoxyl sulfonic acid (DSA), is incorporated into two different hydrogel films, agarose and a copolymer of acrylamide and 2-(dimethylamino)ethyl methacrylate (DMAEM) cross-linked with N,N\u27-methylenebisacrylamide. The swelling and contracting of the hydrogels in response to relative humidity alters the polarity of the environment of DSA, stimulating a shift in the emission wavelength. From 0 to 100% RH, acrylamide-DMAEM sensors exhibited a 40 and 15 nm wavelength shift in still air and flowing gas, respectively. Agarose sensors showed a 40 nm wavelength shift from 0 to 100% RH in still air and a 30 nm shift from 0 to 70% RH in flowing gas. Response times for both sensors were 15 min in still air and less than 5 min in flowing gas. The sensing approach is straightforward and cost-effective, yields sensors with characteristics suitable for commercial measurement of RH (i.e., sensitivity, response times, reproducibility), and allows ease of adaptability to specific RH measurement requirements. The results support the potential extension of the method to a wide variety of analytes in the vapor phase and aqueous solution by incorporation of functionalized smart hydrogels. © 2010 American Chemical Society

Increased Stability of Glycol-Terminated Self-Assembled Monolayers for Long-Term Patterned Cell Culture

Self-assembled monolayers (SAMs) are widely used to confine proteins and cells to a pattern to study cellular processes and behavior. To fully explore some of these phenomena, it is necessary to control cell growth and confinement for several weeks. Here, we present a simple method by which protein and cellular confinement to a pattern can be maintained for more than 35 days. This represents a significant increase in pattern stability compared to previous monolayer systems and is achieved using an amide-linked glycol monomer on 50 Å titanium/100 Å gold-coated glass coverslips. In addition, this study provides insight into the method of SAM degradation and excludes interfacial mixing of the monomers and blooming of the adlayer as major mechanisms for SAM degradation