Timothy V. Ratto

Obstructed Diffusion in Phase-Separated Supported Lipid Bilayers: A Combined Atomic Force Microscopy and Fluorescence Recovery after Photobleaching Approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.

Force spectroscopy of the double-tethered concanavalin-A mannose bond.

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.

Domain Growth, Shapes, and Topology in Cationic Lipid Bilayers on Mica by Fluorescence and Atomic Force Microscopy

Domain formation in mica-supported cationic bilayers of dipalmitoyltrimethylammoniumpropane (DPTAP) and dimyristoyltrimethylammoniumpropane (DMTAP), fluorescently doped with an NBD (((7-nitro-2-1, 3-benzoxadiazol-4-yl)amino)caproyl) phospholipid, was investigated with fluorescence microscopy and atomic force microscopy. Heating above the acyl chain melting temperature and cooling to room temperature resulted in nucleation and growth of domains with distinguishable patterns. Fractal patterns were found for DPTAP, whereas DMTAP domains were elongated and triangular with feathery edges. Reducing the cooling rate or probe concentration for DPTAP bilayers resulted in larger, filled-in domains with more rounded edges. However, for DMTAP, cooling rates mainly affected size and only slightly modified domain morphology. In a saline environment, the domains were dark, and the surrounding continuous region was bright and thus contained the fluorescent probe. However, as the salt concentration was decreased, the dark regions percolated (connected), resulting in bright domains. Atomic force microscopy scans along domain edges revealed that the dark regions in fluorescence images were approximately 1.4 nm thicker than the light regions. Additionally, the dark regions were of bilayer thickness, approximately 4 nm. Comparison of these results in bilayers to well-documented behavior in Langmuir monolayers has revealed many similarities (and some differences) and is therefore useful for understanding our observations and identifying possible growth mechanisms that may occur in domain formation in cell membranes or supported membrane systems.

Chemical vapor discrimination using a compact and low-power array of piezoresistive microcantilevers

A compact and low-power microcantilever-based sensor array has been developed and used to detect various chemical vapor analytes. In contrast to earlier micro-electro-mechanical systems (MEMS) array sensors, this device uses the static deflection of piezoresistive cantilevers due to the swelling of glassy polyolefin coatings during sorption of chemical vapors. To maximize the sensor response to a variety of chemical analytes, the polymers are selected based on their Hildebrand solubility parameters to span a wide range of chemical properties. We utilize a novel microcontact spotting method to reproducibly coat a single side of each cantilever in the array with the polymers. To demonstrate the utility of the sensor array we have reproducibly detected 11 chemical vapors, representing a breadth of chemical properties, in real time and over a wide range of vapor concentrations. We also report the detection of the chemical warfare agents (CWAs) VX and sulfur mustard (HD), representing the first published report of CWA vapor detection by a polymer-based, cantilever sensor array. Comparisons of the theoretical polymer/vapor partition coefficient to the experimental cantilever deflection responses show that, while general trends can be reasonably predicted, a simple linear relationship does not exist.

Lipid Domains in Supported Lipid Bilayer for Atomic Force Microscopy

Phase-separated supported lipid bilayers have been widely used to study the phase behavior of multicomponent lipid mixtures. One of the primary advantages of using supported lipid bilayers is that the two-dimensional platform of this model membrane system readily allows lipid-phase separation to be characterized by high-resolution imaging techniques such as atomic force microscopy (AFM). In addition, when supported lipid bilayers have been functionalized with a specific ligand, protein-membrane interactions can also be imaged and characterized through AFM. It has been recently demonstrated that when the technique of vesicle fusion is used to prepare supported lipid bilayers, the thermal history of the vesicles before deposition and the supported lipid bilayers after formation will have significant effects on the final phase-separated domain structures. In this chapter, three methods of vesicle preparations as well as three deposition conditions will be presented. Also, the techniques and strategies of using AFM to image multicomponent phase-separated supported lipid bilayers and protein binding will be discussed.

An analytic model of thermal drift in piezoresistive microcantilever sensors

A closed-form semiempirical model has been developed to understand the physical origins of thermal drift in piezoresistive microcantilever sensors. The two-component model describes both the effects of temperature-related bending and heat dissipation on the piezoresistance. The temperature-related bending component is based on the Euler–Bernoulli theory of elastic deformation applied to a multilayer cantilever. The heat dissipation component is based on energy conservation per unit time for a piezoresistive cantilever in a Wheatstone bridge circuit, representing a balance between electrical power input and heat dissipation into the environment. Conduction and convection are found to be the primary mechanisms of heat transfer, and the dependence of these effects on the thermal conductivity, temperature, and flow rate of the gaseous environment is described. The thermal boundary layer value that defines the length scale of the heat dissipation phenomenon is treated as an empirical fitting parameter. Using t...

Single‐Molecule Approach to Understanding Multivalent Binding Kinetics

Multivalency results from the simultaneous binding of multiple ligands with multiple receptors. Understanding the effect of multivalency on binding kinetics in molecular and cellular systems may aid the development of new types of therapeutics or countermeasures to pathogen infection. Here, we describe a method using single‐molecule dynamic force spectroscopy to determine the binding strength of antibody–protein complexes as a function of binding valency in a direct and simple measurement. We used the atomic force microscope to measure the force required to rupture a single complex formed by the MUC1 protein, a cancer indicator, and therapeutic antibodies that target MUC1. We will show that nanomechanical polymer tethers can be used in a new manner to count the number of biological bonds formed. Mechanical work will disrupt these bonds and can be used to quantify the overall kinetics. This ability to form, count, and dissociate biological bonds with nanomechanical forces provides a powerful method to study the physical laws governing the interactions of biological molecules.

MODEL CELL MEMBRANE SURFACES FOR MEASURING RECEPTOR–LIGAND INTERACTIONS

In this chapter, practical and theoretical issues concerning measurements of receptor–ligand interactions at domain surfaces of model cell membranes are discussed. We begin with background on the existence and function of lipid domains (so-called “raft” structures) in cell membranes. We present supported lipid bilayers as a potential model system for studying lipid domains and receptor–ligand interactions at domain surfaces. Methods are discussed to form domains in supported lipid bilayers that allow them to adopt their “equilibrium” or nonequilibrim microstructure (especially with respect to domain size). This important development, in combination with variation in domain composition, makes it possible to vary a number of parameters that may modulate receptor–ligand interactions at domain surfaces, particularly in the case of multivalent binding. We present force-spectroscopy as a suitable method to quantify receptor–ligand interactions at domain surfaces of supported lipid bilayers, especially in the case of biological interactions involving a shear stress or force. The general principle and theory of dynamic force spectroscopy are set forth, followed by a practical discussion of how to obtain measurements for specific interactions using dynamic force spectroscopy. From a practical standpoint, it is still necessary to determine if the force to pull domain lipids out of a supported lipid bilayer is significantly different from the forces involved with receptor–ligand bond rupture, and the present understanding of the magnitude of these forces is discussed briefly. If it is assumed that forces of receptor–ligand bonds of interest are significantly larger than lipid pullout forces, multivalent receptor–ligand interactions at domain surfaces of supported lipid bilayers could be measured by varying force loading rates.

In Situ Characterization of Surface Evolution on Titanium in Hydrogen Peroxide Containing Solutions

Titanium implants have been used for decades with success in various applications. The characteristics of titanium that allows acceptance in the body are not well defined. It is known that hydrogen peroxide is a chemical species produced during the inflammatory response following implantation. When titanium is exposed to hydrogen peroxide, a Ti-peroxy gel (TiOOH) is formed. Three possible functions of Ti-peroxy gel are: reduction of the inflammatory response through the reduction of hydrogen peroxide and other reactive oxygen species; creation of a favorable surface for calcium phosphate nucleation; and as a transitional layer between the soft tissue and the stiff titanium. These studies utilized atomic force microscopy (AFM) force spectroscopy, electrochemical techniques, Raman spectroscopy, and optical transparency in situ to define kinetic and mechanical properties of Ti-peroxy gel as it forms on titanium during exposure to hydrogen peroxide. Peaks attributed to Ti-peroxy gel were seen to emerge over the course of several hours using in situ Raman spectroscopy. Force-distance curves suggest a layer that thickens with time on the titanium sample surface.

Interaction Between Titanium Implant Surfaces and Hydrogen Peroxide in Biologically Relevant Environments

Titanium was exposed to dilute solutions of hydrogen peroxide (H 2 O 2 ) to better characterize the interaction at the interface between the solution and metal. The intensity of light passing through films of known thickness of titanium on quartz was measured as a function of time in contact with H 2 O 2 in concentrations of 0.3% and 1.0%. An atomic force microscope (AFM) was used to record deflection-distance (force) curves as a probe approached the interface of titanium in contact with solution containing 0.3% of H 2 O 2 . The interaction layer measured using AFM techniques was much greater than the thickness of the titanium films used in this study. Raman spectroscopy taken during interaction shows the emergence of a Ti-peroxy gel and titania after 2 hours in contact with 0.3% H 2 O 2 solution.