Research in the Thomas P. Beebe, Jr. Group

Group members: Back row (l to r): Dr. Yingjie Zhu, Ben Van Tassell, Brian Fitchett, Jennifer McBride, Vladimir Kushnir, Sven Ammermann; Front row: Yu-Shiu Lo, Becky Jachmann, Terra Hansen

My research group focuses on surface chemistry, surface biology, and surface physics. We engage in multi-disciplinary projects that employ state-of-the-art surface analytical tools including scanning tunneling microscopy (STM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), time of flight secondary ion mass spectrometry (TOF-SIMS), and Auger electron spectroscopy (AES). What follows is an overview of the current projects in the Beebe labs and the Surface Analysis Facility.

Projects Described Here

• Studies of Individual Ligand-Receptor Interactions with Atomic Force Microscopy
• Interactions Between Chiral Amino Acids & Peptides
• Surface Chemical Characterization of 2.5-µm Particulates (PM_2.5) from Air Pollution in Salt Lake City Using TOF-SIMS, XPS and FTIR
• Controlled Production of Molecule Corrals on HOPG Using Ion Bombardment
• Growth of Gold, Silicon, and Semi-Conductor Nanostructures
• Surface Characterization of Single Stranded DNA on Glass

Research Projects

Studies of Individual Ligand-Receptor Interactions with Atomic Force Microscopy

Yu-Shiu Lo, Yingjie Zhu, and Vladimir Kushnir

Highly specific interactions between biomolecules, such as ligand-receptor, antibody-antigen, and complementary double stranded DNA, play a primary role in governing molecular recognition processes in numerous biological functions. While thermodynamic binding properties of biomolecular recognition systems are frequently measured, the direct measurement of the forces involved in these biological systems remains relatively unstudied. Atomic force microscopy (AFM), with its high force sensitivity and capability of operation under physiological liquid environments, is well suited for the studies of such biological interactions.

A unique statistical analysis method based on Poisson statistics has been developed in our group [1-4]. This method (Poisson Statistical Analysis) allows one to determine the magnitude of individual bond-rupture forces as well as nonspecific interactions using the AFM. We have the surface chemistry expertise to place well-controlled chemical and biological functionality on the AFM tips and surfaces. Before force measurements are made, all modified surfaces are characterized by various surface-sensitive techniques, including XPS, TOF-SIMS, and contact-angle measurement.

There are several objectives of this project: 1.) To design and characterize biologically-functionalized surfaces and tips for AFM force measurements of the interaction between individual ligand-receptor pairs with the Poisson statistical method; 2.) To explore ligand-receptor bond-rupture forces and their dependence on loading-rate and temperature in order to understand the nature of the unbinding forces measured experimentally; 3.) To extend our studies to other biological recognition systems including complementary nucleotide strands, integrin receptors on cell surfaces, and neurotransmitter-receptor interactions.

Biotin-avidin and biotin-streptavidin interactions, robust prototypes of ligand-receptor recognition, were selected as our initial research systems. The biotin- and avidin-modified surfaces were prepared [5,6] via the essentially irreversible adsorption of a protein "glue", bovine serum albumin (BSA), onto the piranha-cleaned AFM cantilever tips and the glass substrates [7]. The schematic of the modified tip and substrate is shown in Fig. 1. Four spatially-resolved TOF-SIMS ion maps of an avidin-modified AFM cantilever are shown in Fig. 2. From these images and other related experiments, we can be confident of the presence of protein on the AFM tips in a uniform film on the micron length scale.

All AFM force measurements were acquired in neutral phosphate buffered saline solution, because of its similarity to normal physiological conditions. Measurements were made on the biotin-biotin control system and the biotin-avidin and biotin-streptavidin experimental systems (see Figure 1). Several sets of measurements were made, and the mean and variance of each set was determined. The magnitudes of the interaction rupture force (350 ± 30 pN) between biotin-avidin and biotin-streptavidin pairs can be determined from the slope of the linear regression fit of a variance-versus-mean plot using the Poisson AFM method as shown in Fig. 3.

In contrast to equilibrium binding properties, the rupture strengths of weak chemical or biochemical bonds are not constants but instead are dependent upon the rate and duration of force loading. A proper interpretation of an AFM experiment should account for the detailed experimental parameters, including the cantilever stiffness, speed of pulling, and the loading rate. Measuring bond strengths under controlled loading over a range of rates, which E. Evans refers to as “dynamic force spectroscopy”, can be used to study the pronounced features of the energy landscape along the force-driven rupture pathway [8]. For biotin-avidin and biotin-streptavidin systems, dynamic force spectra, which are plots of bond strength vs. log_e(loading rate), have been acquired in a recent biomembrane force probe (BFP) study at force loading rates in the range of 0.05 to 60,000 pN/s [8]. In our group, the dynamic force spectrum of the biotin-streptavidin bond strength in solution was extended from loading rates of ~10⁴ to ~10⁷ pN/s with the AFM [9]. An AFM study of the temperature dependence of ligand-receptor bond-rupture forces, the first to our knowledge, is currently underway.

References

1. Williams, J. M., Taejoon, H. & Beebe, T. P., Jr. Determination of single-bond forces from contact force variances in atomic force microscopy. Langmuir 12, 1291-1295 (1996).
2. Han, T., Williams, J. M. & Beebe, T. P., Jr. Chemical bonds studied with functionalized atomic force microscopy tips. Analytica Chimica Acta 307, 365-376 (1995).
3. Wenzler, L. A. et al. Measurements of single-molecule bond-rupture forces between self-assembled monolayers of organosilanes with the atomic force microscope. Langmuir 13, 3761-3768 (1997).
4. Wenzler, L. A., Moyes, G. L., Harris, J. M. & Beebe, T. P., Jr. Single-molecule bond-rupture force analysis of interactions between AFM tips and substrates modified with organosilanes. Anal. Chem. 69, 2855-2861 (1997).
5. Moy, V. T., Florin, E.-L. & Gaub, H. E. Intermolecular forces and energies between ligands and receptors. Science 266, 257-259 (1994).
6. Lo, Y.-S. et al. Specific interactions between biotin and avidin studied by atomic force microscopy using the Poisson statistical analysis method. Langmuir 15, 1373-1382 (1999).
7. Lo, Y.-S. et al. Organic and inorganic contamination on commercial AFM cantilevers. Langmuir 15, 6522-6526 (1999).
8. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397, 50-53 (1999).
9. Lo, Y.-S. & Beebe, T. P., Jr. Loading-rate dependence of ligand-receptor bond-rupture forces studied by atomic force microscopy. Langmuir, 17(12) (2001) 3741-3748. View PDF file of paper.

Interactions Between Chiral Amino Acids & Peptides

Yu-Shiu Lo, Vladimir Kushnir

Chirality plays an essential role in the chemistry of life. A majority of biological ligands are chiral. Numerous receptors and enzymes, which are chiral and made of chiral constituents, interact specifically with only one of the enantiomers of a ligand. In pharmacology, the physiological effects and thus the potencies of drugs in different chiral forms may differ significantly. Therefore, the development of techniques with chiral discrimination capability is of great interest in biochemical and pharmaceutical industries.

In our preliminary investigation cysteine (Cys) was chosen because its free thiol group is favorable for surface modification, via gold–thiol bonding (See Figure 4). However, our analysis revealed no significant distinction between the interaction of (L, L), (D, D), and (L, D) Cysteine (See Figure 5). This may be due to the fact that the differences in the non-covalent interactions between unlike cysteine conformations are too weak to be distinguished using the methods used in this study. The hypothesis is backed up by the fact that cysteine is a relatively small molecule, without any strongly polar groups.

Further study of non-covalent interactions of amino acids similar to cysteine would likely give results similar to what was observed in the experiments conducted. Future AFM studies into interactions of chiral biomolecules will focus on polar charged amino acids, and relatively small, configurationally modified biomolecules, like peptide hormones. Non-covalent interactions in the proposed systems would theoretically be much stronger than with small amino acids like cysteine.

Surface Chemical Characterization of 2.5-µm Particulates (PM_2.5)

from Air Pollution in Salt Lake City Using TOF-SIMS, XPS and FTIR

Yingjie Zhu

Chemical analyses of airborne particulate matter with a diameter of 2.5 µm (PM_2.5) are of great importance because of the health effects that PM_2.5 has caused. PM_2.5 can contain a wide variety of toxic metals and toxic organic compounds such as polycyclic organic matter (POM). Many POM species are mutagenic and/or carcinogenic and are thought to damage DNA. The US Environmental Protection Agency (EPA) set a particulate matter standard of diameter < 10 µm (PM₁₀) in 1982. Recent research suggests that there is no safe level of PM₁₀ in the atmosphere to which we may be exposed. For these reasons, the EPA has extended its regulations for particulate matter emissions from the PM₁₀ down to a more stringent PM_2.5. This drives the need for an understanding of the chemistry associated with smaller particulate sizes, and it presents some very interesting surface analytical challenges that our group is well suited to address.

PM_2.5 collected in Salt Lake City is studied using TOF-SIMS, XPS and FTIR (Fourier Transform Infrared Spectroscopy). The high spatial resolution and surface-sensitive characteristics of TOF-SIMS enable the surfaces of individual particulates to be analyzed.

The high mass resolution of TOF-SIMS allows good separation of signals from different chemical species with the same nominal mass (See Figure 6). The extremely high sensitivity of TOF-SIMS also makes the detection of trace elements present in PM_2.5 possible. A number of aromatic compounds (See Figure 7) as well as uranium-containing species in SLC PM_2.5 (See Figure 8) were identified by TOF-SIMS. XPS and FTIR were used to complement TOF-SIMS analysis, and the results are consistent with those from TOF-SIMS. Fig. 9 shows the positive-ion images for some metallic ions detected by TOF-SIMS in Salt Lake City PM_2.5. These images show the lateral distribution, within a randomly chosen 60 ´ 60 mm area of the filter, of specific chemical species on the surface of particulates. One can see that the average size of particulates is about 2 or 3 mm. Some particulates seem larger (see arrow 1) due to the aggregation of several smaller particulates. These chemically and spatially resolved ion images also provide chemical composition information about each individual particulate. Note, for example, that the particle indicated by arrow 2 contains a relatively higher Mg intensity than Na (a particle is not present with high signal-to-noise ratio in the Na image). This particle also contains Al, Si, K, Ca, Fe and Li. The particle indicated by arrow 3 appears to be a small iron particle with relatively low intensity for all other displayed masses. Other individual differences can be ascertained by careful inspection. We are exploring more sophisticated data processing and image analysis tools for images such as Fig. 9, and these will be discussed in a forthcoming publication.

Zhu, Yingjie; Olson, N.; Beebe, T. P., Jr. Surface Chemical Characterization of 2.5-mm Particulates (PM2.5) from Air Pollution in Salt Lake City Using TOF-SIMS, XPS, and FTIR. In press, Env. Sci & Technol. View/Download PDF file.

Molecule corrals are nanometer-sized pits that can be formed with a high degree of control on the basal plane of highly oriented pyrolytic graphite (HOPG). They have a number of unique features that make them useful for several purposes: (1) They can be produced by a simple bench top oxidative process in an oven operating in the ambient air, producing CO₂ (g); (2) They can be produced with a diameter from one nanometer to several microns by varying the total reaction time used to produce them; (3) They are produced in an inherently parallel process that produces ~10⁹ nearly identical corrals covering the entire surface; (4) They can be produced with an extremely narrow size distribution, or as several sets of corrals, each with an extremely narrow size distribution; (5) They can be produced with a density that ranges from less than 1 per mm² to more than 100 per mm²; (6) They can be produced in both monolayer-deep and multilayer-deep, flat-bottomed versions; (7) Once formed, they can be used in a range of diverse applications and fundamental studies. For example, they can be used to contain and isolate small ensembles of molecules from those on a surrounding terrace, as shown in Figure 10, a function which originally led us to select the name “molecule corrals.”

Controlled surface modification of highly oriented pyrolytic graphite (HOPG) was achieved by bombardment of HOPG with Cs⁺ ions at various energies ranging from 0.24 to 10-keV and at various dose densities. The surface chemistry of HOPG bombarded with energetic Cs⁺ ions was studied using the combined surface analysis techniques of TOF-SIMS, XPS, and STM. The experimental findings suggest that cesium implanted into HOPG exists in an oxidized state, i.e., in a form of cesium oxide. The Cs⁺ bombardment of HOPG enhances oxygen adsorption due to both an increase in dissociative adsorption of oxygen at defect sites produced by Cs⁺ ion bombardment and by the formation of cesium oxide. The surface coverage of cesium on HOPG increases linearly with increasing Cs⁺ dose density, and decreases rapidly with increasing Cs⁺ bombardment energy due to cesium implantation below the surface. The thermal stability of cesium on the HOPG surface shows a complex behavior due to the combined effect of diffusion of Cs⁺ to, and desorption of cesium from, the HOPG surface at elevated temperatures.

The controlled molecule corral pit production by the thermal etching of Cs⁺-ion-bombarded HOPG was achieved and studied. The pit (i.e. “molecule corral”) density, pit yield and pit depth can be accurately controlled by varying Cs⁺ dose density and bombardment energy, as seen below in Figures 11 and 12.

200nm

The formation of defects induced by energetic ion bombardment of HOPG provides a promising method for the production of molecule corrals in the control of pit density, pit spatial distribution, and pit depth. Controlled pit production on ion-bombarded HOPG has promising potential applications both scientifically and technologically.

References

Patrick, D. L., Cee, V. J. and Beebe, T. P., Jr., "Molecule corrals" for studies of monolayer organic films. Science, 1994. 265: p. 231-4.

Patrick, D. L. and Beebe, T. P., Jr., Substrate defects and variations in interfacial ordering of monolayer molecular films on graphite. Langmuir, 1994. 10(1): p. 298-302.

Patrick, D. L., Cee, V. J. and Beebe, T. P., Jr., Mechanism of molecular ordering in monolayer liquid crystal films. J. Phys. Chem., 1996. 100: p. 8478-8481.

Patrick, D. L., Cee, V. J., Morse, M. D. and Beebe, T. P., Jr., Nanometer-Scale Aspects of Molecular Ordering in Nanocrystalline Domains at a Solid Interface: The Role of Liquid Crystal-Surface Interactions Studied by STM and Molecule Corrals. J. Phys. Chem. B, 1999. 103(39): p. 8328-8336.

Patrick, D. L., Cee, V. J., Purcell, T. J. and Beebe, T. P., Jr., Defect Pinning in Monolayer Films by Highly controlled graphite defects: molecule corrals. Langmuir, 1996. 12(7): p. 1830-1835.

Stevens, F., Buehner, D. and Beebe, T. P., Jr., Ordering of Adsorbed Organic Monolayers Confined in Molecule Corrals During Scanning Tunneling Microscopy Observation. J. Phys. Chem. B, 1997. 101: p. 6491-6496.

Stevens, F. and Beebe, T. P., Jr., Kinetics of Graphite Oxidation: Monolayer and Multilayer Etch Pits in HOPG Studied by STM. J. Phys. Chem. B., 1998. 102(52): p. 10799-10804.

Stevens, F. and Beebe, T. P., Jr., Computer modeling of graphite oxidation: differences between monolayer and multilayer etching. Comput. Chem., 1999. 23(2): p. 174-183.

Growth of Gold, Silicon, and other Nanostructures

Jennifer McBride, Ben Van Tassell, Becky Jachmann, Brian Fitchett, and Yingjie Zhu.

Nanometer-sized clusters of metallic and semiconductor atoms have been widely investigated due to the size-dependent structural and electronic properties that differ from those of the bulk metal, and from those of isolated single atoms. These nanostructures offer novel ideas for applications in catalysis, microelectronics, sensors, biological interfaces, and advanced materials design. By confining or growing nanometer-sized structures, interesting properties due to quantum confinement effects can be observed. “Molecule corrals,” monolayer-deep etch pits which form on the basal plane of graphite (see image at right and previous section), have been used in a number of applications and investigations in our group. We have combined vacuum evaporation of metals and semiconductors (silicon) with “molecule corrals” and have used these corrals as templates for the formation of metal and semiconductor nanostructures.

The goals of this work are to further characterize the gold nanostructures and their equilibrium three-dimensional structure after annealing at various temperatures. In addition, transfer of the gold nanostructures is being investigated, and scanning tunneling spectroscopy (STS) is being employed to investigate the current-voltage and electrical properties of templated nanostructures as a function of height and diameter.

This project involves evaporation of gold and semiconductor materials onto molecule corral templates on the surface of graphite. By varying the ratio of the template diameter to the amount of material deposited, we have observed the formation of three distinct types of nanostructures: rings, disks, and flat-topped mesas, as seen below for gold in Figure 13.

The gold and silicon nanostructures were imaged using a home-built scanning tunneling microscope operated under ambient conditions. The total amount of surface coverage by the material gold on the graphite surface compromising the nanostructures was determined using x-ray photoelectron spectroscopy (XPS). XPS is also being used to evaluate the degree of transfer of nanostructures from the graphite template surface to a non-conductive glass substrate, as well as to evaluate the effectiveness of encapsulation of the nanostructures within a polymer matrix. Atomic force microscopy (AFM) will also be used in the future to examine the immobilized nanostructures on other substrates.

An exciting new set of experiments involves deposition of semiconductor materials onto the graphite template surfaces. We are currently investigating the use of silicon and gallium arsenide (GaAs) as materials for the formation of nanostructures. These materials are extremely exciting for their nanoscale properties, and their potential use in future applications.

Reference

McBride, J. D., Van Tassell, B., Jachmann, R. C. and Beebe, T. P., Jr., Molecule Corrals as Templates for Metal Nanostructures. J. Phys. Chem., in press. <<link to PDF file>>

Surface Characterization of Single Stranded DNA on Glass

This project is motivated by the need to understand the modes of attachment for DNA and related molecules on surfaces such as glass. The exciting field of "DNA Chips" requires an in-depth understanding of the covalent attachment of DNA, including strategies for improving and maximizing the bonding. In addition, this chemical system provides some interesting test surfaces and structures with which to test surface-sensitive analytical methods such as XPS, TOF-SIMS, and AFM. These methods have not generally been applied in great detail to biological surfaces, or to hybrid materials consisting of a biological-inorganic interface.

The goals of this work are to fully characterize the covalent attachment of short DNA oligomers to glass, the optimization of attachment schemes, and the possible development of new schemes.

This project involves immobilizing DNA with a 5'-amine linker onto glass microscope slides terminated by an aldehyde self-assembled monolayer. Below is a general reaction scheme for the chemistry that is thought to be involved in this process:

The progress and extent of these reaction steps is being followed, studied and optimized by surface-sensitive analytical methods such as XPS, TOF-SIMS and AFM. From the resulting spectra, and advanced data-processing chemometric tools, we hope to be able to identify diagnostic signals that can be used to distinguish different sequences of the attached DNA for the purpose of determining the structure of short oligonucleotides for which the sequence is known. The DNA therefore provides an interesting test sample with which to explore the surface-sensitive information provided by XPS and TOF-SIMS.

Living in Salt Lake City

One of the most unique features of living in Salt Lake City is the close proximity of many outdoor activities. A twenty-minute walk from campus will place you in the foothills of the mighty Wasatch Mountains. Mill Creek Canyon, Big Cottonwood Canyon, and Little Cottonwood Canyon are all within a 15-30 minute drive from campus. Recreational opportunities include backpacking, mountain biking, climbing, day hiking, camping, etc. Of course, Utah is known for the “best snow on earth,” and winters expand these opportunities to include downhill/backcountry/cross-country skiing, snowboarding, and winter mountaineering. Southern Utah, famous for its many national parks, is easily within a half-day drive from Salt Lake City. Bryce Canyon, Zion, Capitol Reef, Arches, and Canyonlands National Park provide some of the most awe-inspiring geography in the world. For those interested in cultural recreation, Salt Lake City offers operas, ballets, symphonies, and many theatrical productions, as well as numerous restaurants and shops. Salt Lake City is home not only to the Utah Jazz, but also to professional baseball, soccer, and hockey teams. There are various clubs around town that provide the listener with all musical genres. There’s no doubt about it, Salt Lake City is a great place to live!

Surface Analysis Facility

Professor Beebe established and manages the University’s Surface Analysis Facility, described below. A more detailed description can be found on the web at

http://www.chem.utah.edu/chemistry/facilities/surface/index.html

X-ray Photoelectron Spectroscopy. XPS is a widely used method of determining the chemical composition of a surface. X-rays impinge upon a sample and ionize atoms, releasing core-level photoelectrons. The escaping photoelectron's kinetic energy limits the depth from which it can emerge, giving XPS its high surface sensitivity and sampling depth of a few nanometers. Photoelectrons are collected and analyzed by the instrument to produce a spectrum of emission intensity versus electron binding energy. Peak areas at nominal binding energies can be used to quantify elemental composition, and small shifts in these binding energies (chemical shifts) provide powerful information about sample chemical states and short-range chemistry. XPS is suitable for the analysis of conductors and insulators such as polymers.

The instrument can also rapidly collect XP images with a lateral resolution of approximately 1 micron and sufficient spectral resolution to discern, for example, carbidic and graphitic carbon domains in a few minutes of acquisition time. All image pixels are collected in parallel with a large time advantage over other imaging methods. The combination electrostatic-magnetic lens system provides higher spatial resolution than an electrostatic-only system. Small-spot spectral analysis down to approximately 20 microns is available.

The multi-technique system includes a LaB₆ electron gun as an excitation source for Auger. This gun can also be used for 100-nm-resolution SEM. In Auger, an incident primary electron creates an excited ion near the surface which decays by the emission of a secondary Auger electron, whose kinetic energy is measured. As in photoelectron spectroscopy, the escaping Auger electron's kinetic energy limits the depth from which it can emerge, giving AES its high surface sensitivity and few nanometer sampling depth. Auger electron spectra can be acquired from a selected area mapped out in an SEM image of the sample (e.g., from within a rectangle, along a line, or at points; at right an AFM cantilever is seen poised above a test grid; three analysis points are selected). Auger images or maps can also be generated for specific elements with approximately 100-nm resolution. Auger finds its greatest strengths in the analysis of inorganic materials not susceptible to electron-beam damage.

Time of Flight Secondary Ion Mass Spectrometry. The Facility now houses the technique of time-of-flight secondary-ion mass spectrometry, with the Cameca/ION-TOF TOF-SIMS IV system. Support was derived from the NSF (DMR-9724307) and the University. ToF-SIMS is a surface sensitive technique used to probe a material's long-range chemical structure through the mass spectral analysis of desorbed molecules and molecular fragments. An incident primary ion blasts molecular fragments from the sample surface; positive or negative secondary ions are collected from these fragments and their mass is determined by flight time to a detector. Several operational modes are now available in the facility:

• static SIMS mode - very low incident ion doses are used to probe the molecular structure and long-range chemistry of surfaces; the low ion dose ensures that intrinsic surface chemistry, and not ion-induced chemistry, is being probed; static SIMS is highly complementary to XPS for monolayer and polymer surface analysis of organic materials.

• dynamic SIMS mode - high incident ion doses are used to sputter off surface layers and probe elemental composition as a function of depth into a sample; all molecular information is scrambled; quantitation and sensitivity can be as high as parts per billion in some cases; dynamic SIMS is useful for inorganic materials and impurity analysis.

• imaging SIMS mode - a focused ion beam can be rastered over a surface to collect static SIMS spectra as a function of lateral position; resolution better than 100 nm can be achieved with very high mass resolution (m/Dm > 10,000).

• SEM mode - a focused ion beam (sub-50-nm) can be rastered over a surface while secondary electrons are collected, as in a conventional SEM. This mode can be used to locate features of interest.