The ultimate goal of this research is to establish a novel assay for the examination of individual proteins in function. The possibility of exploring living cells from nanometer scale to micrometer scale opens up prospective areas in many aspects of cell biology. The effort is four-fold:

a. Imaging of proteins at single molecule level

As the first step toward imaging of proteins on living cells, we achieved high resolution images of various proteins, including cholera toxin B-oligomer, Human IgG antibody, and ferritin on model substrates in buffer media. We also performed imaging on living cells, PC12 neural cells (~ 10 ?m in size), from microscopic scale (single cell level) to nanoscopic level (individual proteins). Images were also achieved on other types of cells, such as HeLa cancer cells and mouse embryonic fibroblast. In future, we will also examine human embryonic stem cells as part of the work funded by NIH.

b. Ligand-receptor interactions and force mapping

Topographic imaging alone can’t tell the species of a protein. Protein species can be identified by its specific interaction with the affinity reagent, usually the ligand or the antibody. We applied surface chemistry for cross-linking ligands / antibodies to an AFM tip. The adhesion force measured between the modified tip and the cell membrane proteins in media on a substrate (either a model substrate or living cell) provided quantitative measure of the specific interaction. When adhesion forces were measured pixel by pixel while the tip scanned on a protein-containing surface, an affinity map was achieved, and the distribution of the protein was revealed. For instance, we used a nerve growth factor (NGF) modified AFM tip to perform force mapping on a living PC12 cell. Distribution of NGF-receptor, TrkA, on the cell was achieved. With such a probe, we found that TrkA tends to associate to each other, forming oligomers on the cell. Such unique protein association may provide the environment for effective NGF-TrkA binding, and consequently the effective function of NGF in neural cells. Similar study was also performed to examine the distribution of cholera toxin B-oligomer and Human IgG on model substrates. We will apply the same method to examine the cell membrane proteins of human embryonic stem cells, which holds great promise to many human diseases.

c. Synthesis and characterization of photo-cleavable cross-linkers

In the previous studies, proteins were permanently cross-linked to an AFM tip. Such a modified tip greatly hindered the requisition of high resolution images of proteins due to the strong ligand-receptor interaction. We designed and synthesized photo-cleavable cross-linkers to anchor ligands on an AFM tip, so that the probe can be photo-switched between a ligand modified tip and a protein-free tip to complete two individual AFM tasks sequentially at the same local region. A photo-cleavable cross-linker, succinic acid succinimidyl ester 5-thioyloxy-2-nitrobenzyl ester (SSTN), was synthesized in five steps. Steady state spectroscopic studies suggested that upon photo-excitation, SSTN undergoes a clean fragmentation reaction with a quantum yield 0.1. SSTN can covalently anchor protein molecules to a gold coated substrate and the protein can be efficiently (>95%) released upon irradiation without degrading its binding activity. As an improvement, we also synthesized a traceless photo-cleavable cross-linker, di 6 -(3-succinimidyl carbonyloxymethyl-4-nitro-phenoxy)-hexanoic acid disulfide diethanlol ester (SCNE). This cross linker allows the full recovery of the linked protein to its nature form. In future, we will synthesize additional cross-linkers (i) with the ability of linking two proteins at each end of the linker and cleavable with different wavelengths of light, (ii) with the ability of linking proteins with selective orientation, (iii) hybridizing with PEG for longer flexible chains and for reducing the non-specific interactions.

d. Development of two-tier AFM

We applied SSTN for reversible protein immobilization. Thus an AFM tip can be photo-switched between a ligand modified tip and a protein-free tip. Taking advantage of the lateral resolution of AFM, we reproducibly achieved protein (such as BSA, human IgG antibody, protein A, avidin) delivery from a tip to a desired location of a substrate with nanoscale precision upon the photocleavage of SSTN. Therefore the irradiation served as a “remote-controller” to precisely control the timing and location of protein delivery. Subsequently and importantly, we achieved high resolution images of the delivered proteins using the protein-free tip. We also proved that the delivered proteins maintained the binding activity toward the counter-proteins. With such a design, we demonstrated that an AFM tip can accomplish two individual tasks subsequently at the same local area. In future, we will apply this methodology in the study of protein association in living cells. Combined with force mapping method, a ligand modified tip can be used to deliver ligand to its receptor at local region of a cell, and the local cell response can be monitored in real-time. This methodology can be generalized and applied to any cell type. The research is expected to provide a platform to elevate our fundamental understanding of molecular ligand-receptor functions to a new level.

We were the first group to use AFM in the study of Bacillus spore coat. The simple sample preparation in AFM study allows the access to spore surfaces in natural conditions. All spores were provided by Dr. Adam Driks at Loyola University Medical Center. This work has relevance to both the classical problem of how the dormant, highly resistant spore is able to rapidly return to active growth, and the current urgent need for tools to combat anthrax, a result of exposure to B. anthracis spores. In this study, we observed unique ridge structures on Bacillus spore coats, the first observation on free spores. Our results indicate the primary driving force for ridge formation is dehydration when the spore interior reduces in volume during spore formation. Since spore coat is the key structure in spore formation and germination, we examined the roles of a large number of coat proteins on ridge formation using various spore mutants, and found that the formation of ridges is controlled at least in part by some subset of the coat proteins. Parallel studies were also carried out with B. anthracis (Stern strain only) spores. CotO and Cotß proteins were identified to play important roles in the formation of the characteristic coat ridges. In addition, we examined the surface features on the spore outer coats of different species and strains of Bacilli using an AFM. We characterized four species of Bacilli spores and three strains of B. subtilis spores. Besides the characteristic ridges on any given spore, we observed strictly parallel fine rodlets with characteristic dimensions on the nanometer scale on all the spore surfaces. The characteristics of ridges and rodlets differ sufficiently among species and strains to permit species-specific and strain-specific spore identification. Further investigations will be carried out to examine more species and strains and to look into the origin of the rodlet structures.

As a newly initiated project (collaborative with Prof. Stetter’s group), the goal of this research is to develop a new generation of chemical / biological sensors based on nano-manipulation of single wall carbon nanotubes (SWCNTs). Starting with the commercially available CNTs, we developed protocols for effective CNT purification. With a sample under such treatment, we were able to cut an individual CNT by applying high forces to a desired local region using an AFM tip. Successful nano-cleavage of an individual CNT has been reproducibly achieved on various samples with different surface treatments and also on various substrates. A minimum of 5 nm gap can be achieved within mille second. We also demonstrated that an individual CNT bridging two electrodes (1 µm apart) on a MEMS structure was cut by an AFM probe. In future, we will apply MEMS to produce interdigitated microelectrode structures. We will examine the electronic signal in response to chemical vapors and biological molecules in the environments.