We describe a method for the production of nanoelectrodes at the apex of atomic force microscopy (AFM) probes. The nanoelectrodes are formed from single-walled carbon nanotube AFM tips which act as the template for the formation of nanowire tips through sputter coating with metal. Subsequent deposition of a conformal insulating coating, and cutting of the probe end, yields a disk-shaped nanoelectrode at the AFM tip apex whose diameter is defined by the amount of metal deposited. We demonstrate that these probes are capable of high-resolution combined electrochemical and topographical imaging. The flexibility of this approach will allow the fabrication of nanoelectrodes of controllable size and composition, enabling the study of electrochemical activity at the nanoscale.

Tetrahydrobiopterin (H 4 B) is a critical element in

A procedure for the batch microfabrication of scanning electrochemical-atomic force microscopy (SECM-AFM) probes is described. The process yields sharp AFM tips, incorporating a triangular-shaped electrode (base width 1 microm, height 0.65 microm) at the apex. Microfabrication was typically carried out on (1)/(4) 3-in. wafers, yielding 60 probes in each run. The measured spring constant of the probes was in the range 1-1.5 N m(-1). To date, processing has been carried out twice successfully, with an estimated success rate for the fabrication process in excess of 80%, based on field emission-scanning electron microscopy imaging of all probes and current-voltage measurements on a random selection of approximately 30 probes. Steady-state voltammetric measurements for the reduction of Ru(NH(3))(6)(3+) in aqueous solution indicate that the electrode response is well-defined, reproducible, and quantitative, based on a comparison of the experimental diffusion-limited current with finite element simulations of the corresponding mass transport (diffusion) problem. Topographical imaging of a sputtered Au film with the SECM-AFM probes demonstrates lateral resolution comparable to that of conventional Si(3)N(4) AFM probes. Combined electrochemical-topographical imaging studies have been carried out on two model substrates: a 10-microm-diameter disk ultramicroelectrode (UME) and an array of 1-microm-diameter UMEs, spaced 12.5 microm apart (center to center). In both cases, an SECM-AFM probe was first employed to image the topography of the substrates. The tip was then moved back a defined distance from the surface and use to detect Ru(NH(3))(6)(2+) produced at the substrate, biased at a potential to reduce Ru(NH(3))(6)(3+), present in bulk solution, at a diffusion-controlled rate (substrate generation-tip collection mode). These studies establish the success of the batch process for the mass microfabrication of SECM-AFM tips.

The spore-forming, gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Upon infection, germination of endospores occurs during their internalization within the phagocyte, and the ability to survive exposure to antibacterial killing mechanisms, such as O 2 ⅐؊ , NO · , and H 2 O 2 , is a key initial event in the infective process. Macrophages generate NO · from the oxidative metabolism of L-arginine, using an isoform of nitric oxide synthase (NOS 2). Exposure of murine macrophages (RAW264.7 cells) to B. anthracis endospores up-regulated the expression of NOS 2 12 h after exposure, and production of NO · was comparable to that achieved following other bacterial infections. Spore-killing assays demonstrated a NO · -dependent bactericidal response that was significantly decreased in the presence of the NOS 2 inhibitor L-N 6 -(1-iminoethyl)lysine and in L-arginine-depleted media. Interestingly, we also found that B. anthracis bacilli and endospores exhibited arginase activity, possibly competing with host NOS 2 for its substrate, L-arginine. As macrophage-generated NO · is an important pathway in microbial killing, the ability of endospores of B. anthracis to regulate production of this free radical has important implications in the control of B. anthracismediated infection.Endospores of the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, when inhaled and deposited in the lung, are phagocytosed by resident macrophages, where the spore evades host defenses and germinates into the vegetative bacterium (6,13,14). Germination is a prerequisite to replication and dissemination of B. anthracis (2). Unfortunately, mechanisms by which the spore enters the macrophage, germinates, survives therein, and escapes from this phagocytic cell are poorly understood. To study spore-specific interactions, the ⌬gerH mutant of the B. anthracis Sterne 34F2 strain, which is a germination-deficient variant incapable of conversion from spores to bacilli in macrophages, is an appropriate model (34). The use of this strain focuses on distinct spore-macrophage interactions independent of contamination of B. anthracis vegetative bacilli.The virulence of B. anthracis and the efficiency with which it can infect humans via inhalation are reasons the organism and the disease are currently being investigated. B. anthracis spores are comprised of an endospore, containing the infectious portions of the bacterium, and a surrounding exosporium, the outermost layer of the spore, composed of polysaccharides, lipids, and approximately 20 different proteins (27). The exosporium is the primary site of contact for spore interactions with host defenses. The process of sonication has been shown to selectively remove the exosporium while keeping the endospore intact (18). We postulate that spore-macrophage interaction at the exosporium is pivotal to the anthrax disease process.Once inside the...