David C. Turner

Selective adhesion of functional microtubules to patterned silane surfaces

We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces.

Modification of supported lipid membranes by atomic force microscopy

The atomic force microscope (AFM) was used to structurally modify supported lipid bilayers in a controlled quantitative manner. By increasing the force applied by the AFM tip, lipid was removed from the scanned area, leaving a cut through the lipid bilayer. Cuts were repaired with the AFM by scanning the region with a controlled force and driving lipid back into the cut. A slow self-annealing of cuts was also observed.

Diffusion and transfer of antibody proteins from a sugar-based hydrogel

Diffusion of antibody protein from hydrogel films and hydrogel encapsulated in a microcapillary was studied. Thin hydrogel films were formed by crosslinking 6-acryloyl-B-O-methylgalactoside withN,N’-methylene-bis-acrylamide and the diffusive transport of monoclonal antimouse IgG-FITC into and out of the hydrated films was measured. Diffusion coefficients in 2 and 4% crosslinked hydrogel films were measured. The measured diffusion constants determined for IgG in both the 2 and 4% hydrogel films were comparable to the free diffusion of IgG in bulk water (Dmean ∼ 10-7cm2/s). In addition, 2% crosslinked hydrogels were prepared in a capillary tube and the transport of antimouse IgG-FITC into and out of the hydrated hydrogel was measured. Kinetic analysis indicated that the protein transport through the capillary hydrogel was faster than would be expected for a simple diffusion process. Finally, by utilizing the diffusion of antibody from the capillary hydrogel, transfer of antibody to a silica surface was demonstrated. A capillary hydrogel loaded with antimouse IgG-FITC was used to transfer the protein to a silica surface forming a 30-μm spot of antibody, which was imaged using fluorescence microscopy. These results may lead to the development of a nonlithographic method of patterning antibodies on surfaces for use in integrated microimmunosensors.

Use of a repositionable substrate to acquire and compare distinct atomic force microscope images of a field of microtubules

Abstract Many scanning probe microscope (SPM) experiments of interest require removing the sample from the scanning head of the SPM for processing or examination by other techniques and then replacing it in the SPM head and relocating the original sample area. For feature sizes below 1 μm this is often a tedious, if not impossible, task. To resolve this problem we have designed a patterned substrate which can be used with any SPM to relocate sub-micrometer features even after removal, and subsequent replacement of the sample in the scanning head. The use of this device with an atomic force microscope (AFM) is demonstrated by imaging a field of chicken brain microtubules with several different AFM probes and scan modes. Contact, Tapping™, and error-signal mode AFM images were acquired using normal pyramidal tips and Park Scientific Ultralevers. Images of individual tubules were obtained with all three imaging modes, allowing direct comparison with respect to height, width and feature resolution.

Direct enzymatic hydrolysis and patterning of a chemisorbed peptide thin film

We have demonstrated that covalently immobilized enzyme can be used to chemically modify and pattern a chemisorbed peptide film on a solid substrate. The enzyme, alpha- chymotrypsin, was covalently attached to silica and latex spherical beads by glutaraldehyde crosslinking to an amino surface functionality. The fluorescent peptide, suc-ala-ala- phe-AMC (SAAP-AMC) was immobilized to an aminosilane- modified flat silica surface by forming an amide bond to its free carboxylic acid group. SAAP-AMC surfaces were characterized using water wetability, fluorescence spectroscopy and x-ray photoelectron spectroscopy. Upon attack by alpha-chymotrypsin coated beads, the fluorescent AMC group (325 nm ex, 395 nm em) is cleaved from the SAAP- AMC peptide surface and a red-shift occurs in the AMC fluorescence providing a standard marker for enzymatic activity (345 nm ex, 440 nm em). Thus, the signature for alpha-chymotrypsin bead activity against the surface is a reduction in fluorescence intensity from the peptide surface (at 395 nm) and a concomitant increase in fluorescence in the solution above the surface (at 440 nm). Treatment of the SAAP-AMC silica surfaces with alpha-chymotrypsin-beads showed a reduction of the surface fluorescence to background in less than 24 hours, with a corresponding increase of free AMC fluorescence in solution. By restricting the contact region of the beads with the peptide surface we were able to demonstrate chemical patterning of the peptide surfaces.

Monolayer and atomic force microscopy studies of nicotinic acetylcholine receptor films

The surface pressure-area and surface potential-area isotherms for the nicotinic acetylcholine receptor (nAChR) and several lipid-protein monolayers are reported. It was shown that buffer composition and pH affect the film isotherm; the addition of unsaturated lipid and cholesterol to the native protein film contributes to the fluid phase of the monolayer. Langmuir-Blodgett (LB) films of the lipid and lipid-protein monolayers transferred to silicon substrates were imaged using atomic force microscopy (AFM). The AFM images of lipid films show regular structure in the lipid layer and indicate protein aggregates in the AChR-lipid LB films, with feature sizes corresponding to the lipid and receptor molecular dimensions.

A crystallographic molecular lattice builder applied to model lipid bilayers

It is often desirable for noncrystallographers to generate graphical models of three-dimensional crystal structures based on published coordinates of the atoms that make up the crystallographic unit cells. This type of visualization is particularly important for small-molecule crystals, such as lipid crystals, where one may be interested in investigating interactions between the individual molecules in addition to their conformations. BILAYER BUILDER is a program that generates a portion of the entire crystal structure from the coordinates of the molecules in a single unit cell. It gives users of small desktop computers, such as the Apple Macintosh, the capability to generate and examine model crystal structures with a molecular graphics display program. BILAYER BUILDER stores the crystal coordinates in a Brookhaven Protein Data Bank file format for possible use in a variety of applications on many different computers. Initially, it was written for use with lipid crystals and bilayers but may be used for building an assortment of molecular crystals.

Tools for Molecular Graphics Depictions of Lipid Structures

We have developed several tools for the molecular graphics depiction of lipids and their microstructures. These include: 1) NanoVision, a molecular visualization program for Macintosh personal computers; 2) NRLipid, a HyperCard database of lipid structural information; 3) BILAYER BUILDER, a program for constructing models of lipid bilayers from crystallographic data; and 4) Ribbon Representation, an adaptation for the depiction of subtle acyl chain structure of a display tool commonly used for proteins.