AVS2013 Session BI+NL+NS+SS-ThM: Bio/Nano Interfaces
Thursday, October 31, 2013 8:20 AM in 201 B
BI+NL+NS+SS-ThM-2 Utility of Lipid Membranes Assembled on Nanoparticles for Measuring Protein Membrane Interactions
Scott Reed (University of Colorado Denver)
Nanoparticles (NPs) provide a well-defined template for preparing supported lipid membranes with controlled curvature. We have coated supported lipid bilayers and hybrid membranes on silica and gold nanoparticles. The localized surface plasmon resonance (LSPR) of gold NPs can be used to monitor the assembly of lipid layers on NPs and to monitor protein lipid interactions. The gold LSPR is very sensitive to the immediate surroundings of the nanoparticle surface and therefore provides a method to monitor the coating of lipids and subsequent conversion of a supported bilayer to a hybrid membrane after the addition of hydrophobic alkanethiols. We demonstrate that both long chain (decanethiol) and short chain (propanethiol) anchors are able to form hybrid membranes and that these membranes allow for LSPR based detection of protein binding events at the membrane surface.
While many materials have been used as membrane supports, there are unmet needs in the development of membrane mimics and it remains challenging to monitor the coating process and to control the curvature of a membrane. Recent work has demonstrated that quantum dots, silica NPs, and gold NPs can be used as templates for membranes providing an opportunity to control curvature. Here, we have exploited the local refractive index sensitivity of the gold LSPR to observe the process of lipid-coating, structural rearrangement of supported membranes into hybrid membranes, and finally the binding of protein. The introduction of phosphatidylcholine (PC) to the gold NPs results in a rapid binding evidenced by a change in the wavelength of the LSPR, however, the interaction with gold is weak and the gold is not completely covered by the lipid. By adding a hydrophobic alkanethiol anchoring group, the lipids bind closer to the gold NP surface resulting in increased stability. This stability is achieved at different concentrations for short and long hydrophobic chains. When propanethiol was used it was possible to destabilize and remove the lipid coating by adding the hydrophilic thiol, beta-mercaptoethanol. This loss of membrane is observed through changes to the LSPR and increased permeability of the membrane to ions.
BI+NL+NS+SS-ThM-3 Primitive Osmosensing by Phospholipid Membranes
Kamila Oglecka (Nanyang Technological University, Singapore); Jeremy Sanborn, Doug Gettel (University of California, Davis); Rachel Kraut, Bo Liedberg (Nanyang Technological University, Singapore); Atul Parikh (University of California, Davis)
This talk describes experimental observations of the response of multicomponent vesicles to osmotic gradients. We find that giant vesicles, consisting of phase separating lipid mixtures, immersed in hypertonic bath exhibit a Rayleigh-Plateu like pearling instability paving for elemental mechanical process of vesicular self-reproduction. When immersed in hypotonic bath, however, the response of giant vesicles comprise an unusual transitory state, characterized by damped, periodic oscillations between a microscopically phase-separated state and a uniform one. We find that this unusual oscillatory phase separation is synchronized with the cyclical patterns of membrane tension and poration, producing swell-burst cycles. Swelling, which is caused by the influx of water, raises membrane tension, thus promoting the appearance of microscopic domains. Bursting, which facilitates solute leakage, relaxes the membrane tension, breaking up large domains into those below the optical limit. This autonomous self-regulatory response – in which an external osmotic perturbation is managed by a co-ordinated and cyclical sequence of simple physical mechanisms. These mechanisms allow vesicles to sense (by domain formation) and regulate (by solute efflux) osmotic differences across their compartmental boundaries in a negative feedback loop producing a primitive form of a quasi-homeostatic regulation in a synthetic system, generated from simple components, namely, water, osmolytes, and lipids.
BI+NL+NS+SS-ThM-5 Ultrathin Poly(ethylene glycol) Films as Flexible Platform for Plasmonics and Lithography and as Precursors for Free-Standing Nanomembranes
Nikolays Meyerbröker, Michael Zharnikov (University of Heidelberg, Germany)
We present a novel approach to prepare ultrathin, biocompatible films based on cross-linking of multi-functionalized, star-branched poly(ethylene glycols) (STAR-PEGs) with tunable film thicknesses of 4 – 200 nm. A two-component mixture of amine- and epoxy-terminated four-arm STAR-PEGs was spin-coated on a flat substrate and cross-linked chemically by gentle heating, resulting in a stable, hydrogel-like film with a density close to that of bulk PEG material. The films revealed pronounced swelling behavior, which was fully reversible and could be precisely controlled. Additionally, they provided a high affinity to citrate-stabilized gold nanoparticles (AuNP) that could be adsorbed with high densities into the PEG matrix from an aqueous solution. These novel PEG/AuNP composite films offer interesting and potentially useful optical properties. Controlling the accessibility, swelling behavior, and biorepulsive properties of the PEG films lithographically, we prepared nanocomposite patterns of metal nanoparticles and fluorophores imbedded into the PEG matrix as well as protein-affinity patterns in protein-repelling background. Further, using electron beam lithography, we succeeded to fabricate wettability patterns and to sculpture complex 3D microstructures on the PEG basis. Finally, we demonstrated that the PEG films can be separated from the substrate and exist as ultrathin, biocompatible, free-standing membranes. These membranes possess high stability and exceptional elasticity. They can be used in transmission electron microscopy experiments on sensitive biological targets and as a new type of support for the characterization of nanoparticles.
BI+NL+NS+SS-ThM-6 Miniaturized Localized Surface Plasmon Resonsance Sensing with Single Nanoparticle Arrays
Si Chen, Mikael Svedendhal, Tomasz Antosiewicz, Mikael Käll (Chalmers University of Technology, Sweden)
Ultrasensitive biosensing is one of the main driving forces behind the dynamic research field of plasmonics. I will show that the sensitivity of single metal nanoparticle plasmon spectroscopy can be greatly enhanced by enzymatic amplification of the refractive index footprint of individual protein molecules, so called plasmon-enhanced ELISA. The technique, which is based on generation of an optically dense precipitate catalyzed by horseradish peroxidase at the metal surface, allowed for colorimetric analysis of ultralow molecular surface coverages with a limit of detection approaching the single molecule limit. In addition I will show that by combining large arrays of well-separated gold nanoparticles fabricated by electron beam lithography (EBL) with hyper spectral imaging, spectral responses of up to 700 LSPR particles can be simultaneously studied. This allows us to obtain enough statistical significant number of spectra to further study the inhomogeneous broadening of the sensing properties of individual particles. This includes how variation in electric field enhancement over the surface of a single particle and variation in size and morphology of the enzymatic precipitate could affect the uncertainty in determining the number of enzyme molecules per particle. By combining the electromagnetic simulations with the measurements we could conclude that main sources of uncertainty come from variations in sensitivity across the surface of individual particles and between different particles. There is also a considerable uncertainty in the actual precipitate morphology produced by individual enzyme molecules. I will also discuss the possible improvement that can be done to achieve digital responses from the enzymatic amplified single particle sensing.
|10:00 AM||BREAK - Complimentary Coffee in Exhibit Hall|
BI+NL+NS+SS-ThM-9 Design of Nanoscale Bionterfaces by Self-Assembly of Genetically Encoded Peptide Polymers
Ashutosh Chilkoti (Duke University)
This talk will cover work in my laboratory over the past decade on the self-assembly of genetically encoded stimulus responsive elastin-like polypeptides (ELPs). We have exploited ELPs to create stimulus responsive nanostructures via three approaches. In the first approach, we have designed diblock ELPs with two ELP blocks with different hydrophobicity’s that self-assemble into spherical micelles with an increase in temperature about the critical micellization temperature of the diblock polymer. Building on this architecture, we have incorporated histidine residues in the hydrophobic block to create a diblock ELP that self-assembles into spherical micelles with an increase in temperature, while a small drop in pH from 7.4 to 6.4 leads to micelle disassembly. In a second –chemical attachment triggered self-assembly– approach, we have shown that the attachment of multiple copies of small molecule hydrophobes to the multiple cysteine (C) residues of an ELP with the sequence (VPGXG)n(CGG)8 can drive their self-assembly into spherical micelles. In a third approach, we replace the Cys (C) with W, Y, or F, and find that oddly, this leads to the formation of stimulus responsive worms and vesicles depending on the specific residue. These are the first examples of stimulus responsive worms and vesicles in peptide polymers.
BI+NL+NS+SS-ThM-11 Label-Free Mapping of Protein/Peptide Interactions in Complex Arrays Using Core/Shell Nanoparticle-Based Biosensors
HaciOsman Guvenc (University of Heidelberg, Germany); Christopher Schirwitz, Frank Breitling (Karlsruhe Institute of Technology, Germany); FrankRalf Bischoff (German Cancer Research Center Heidelberg, Germany); Alexander Nesterov-Mueller (Karlsruhe Institute of Technology, Germany); Volker Stadler (PEPperPRINT GmbH, Heidelberg, Germany); Jenny Wagner, Reiner Dahint (University of Heidelberg, Germany)
The detailed analysis of biospecific interactions is of crucial importance in biomedicine, biotechnology, and pharmacology.Important applications range from medical diagnosis and drug development to the screening of the human genome and proteome. In recent years, array concepts have become very popular and powerful tools to allow for highly parallel, rapid identification of binding events. These arrays contain a multitude of different probe molecules immobilized at specific locations of an underlying substrate.
Interaction analysis is usually facilitated by labelling the potential binding partners with additional markers. Today, many of such techniques are well-established yielding considerably low detection limits and high lateral resolution. Yet, they suffer from the fact that labelling procedures are usually costly and time-consuming, that the labelling efficiency needs to be properly controlled for quantitative analysis, and that the marker itself can affect the original functionality of the molecules being studied. Moreover, the detection of low-affinity binding events is often hampered as additional washing steps and (bio)chemical reactions are required in-between the interaction and detection processes. To overcome those obstacles, strong efforts have been made to establish label-free detection schemes for interaction analysis. However, marker-free, sensitive readout of high-density arrays is still a technological challenge.Here we report recent experiments on the label-free detection of protein/peptide interactions in complex arrays based on surface plasmon imaging with core-shell nanoparticle monolayers. Upon reflection of white light, these films exhibit a pronounced extinction spectrum which shifts to higher wavelength upon molecule binding, thus, providing a simple, sensitive and label-free detection mechanism. Variants of HA (human influenza hemagglutinin A) and FLAG epitopes with permuted amino acid sequence are synthesized in array format by means of combinatorial chemistry using a novel laser printing approach. After array preparation, the pattern is cleaved from the carrier and transferred to a biosensor surface consisting of core-shell nanoparticle films. By this means, the arrays are purified from synthesis artefacts caused by incomplete coupling reactions in the synthesis without the loss of spatial resolution. The interaction of the different peptides with their respective antibodies is quantified by the wavelength shift observed for individual peptide spots and compared to fluorescence-based interaction analysis. Based on the data we conclude on relevant amino acid sequences for an efficient antibody/epitope binding.
BI+NL+NS+SS-ThM-12 Graphene for Biosensing and Surface Functionalization
Paul Sheehan (Naval Research Laboratory); Rory Stine (Nova Research); Shawn Mulvaney, Jeremy Robinson, Cy Tamanaha (Naval Research Laboratory)
Graphene, a one-atom thick sheet of sp2 carbon, offers many intriguing possibilities in the field of molecular sensing. Its unique combination of large areas with nanometer thickness and high electrical conductivity could enable small scale device sensitivity with large scale production methods. A major benefit of using graphene is the large toolbox of well-established chemistries for incorporating chemical functionalities or specific recognition elements at the device surface. Here, we will discuss our efforts to develop graphene-based biological field-effect transistors (BioFETs), which offer sensitivity comparable to sensors made with other nanoscale materials (carbon nanotubes, nanowires), but with greatly simplified production methods common in the semiconductor industry. Devices utilizing both graphene and graphene oxide will be covered, and surface spectroscopic studies of the material modification will be discussed. Successful results for the detection of specific DNA hybridization using graphene BioFETs will also be presented. We will further discuss our efforts to use graphene as a biofunctionalized interface for a number of materials, from polymers to dielectrics to semiconductors, of interest to the biosensing community. Graphene’s ultrathin nature allows its inclusion in more traditional sensing platforms as a non-intrusive functionalization layer, discreetly lending its chemical flexibility to other, more inert materials without significantly impacting the sensing device.