Advances in endotoxin analysis.

The outer membrane of gram-negative bacteria is primarily composed of lipopolysaccharide (LPS). In addition to protection, LPS defines the distinct serogroups used to identify bacteria specifically. Furthermore, LPS also act as highly potent stimulators of innate immune cells, a phenomenon essential to understanding pathogen invasion in the body. The complex multi-step process of LPS binding to cells involves several binding partners, including LPS binding protein (LBP), CD14 in both membrane-bound and soluble forms, membrane protein MD-2, and toll-like receptor 4 (TLR4). Once these pathways are activated, pro-inflammatory cytokines are eventually expressed. These binding events are also affected by the presence of monomeric or aggregated LPS. Traditional techniques to detect LPS include the rabbit pyrogen test, the monocyte activation test and Limulus-based tests. Modern approaches are based on protein, antibodies or aptamer binding. Recently, novel techniques including electrochemical methods, HPLC, quartz crystal microbalance (QCM), and molecular imprinting have been developed. These approaches often use nanomaterials such as gold nanoparticles, quantum dots, nanotubes, and magnetic nanoparticles. This chapter reviews current developments in endotoxin detection with a focus on modern novel techniques that use various sensing components, ranging from natural biomolecules to synthetic materials. Highly integrated and miniaturized commercial endotoxin detection devices offer a variety of options as the scientific and technologic revolution proceeds.

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold.

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, drug loading and release, and the development of catalysts have unquestionably accelerated the usage of nanoporous gold (NPG)-based nanomaterials in research and development. This article describes the process of the generation of hierarchical bimodal nanoporous gold (hb-NPG) by employing a step-wise procedure involving electrochemical alloying, chemical dealloying techniques, and annealing to create both macro- and mesopores. This is done to improve the utility of NPG by creating a bicontinuous solid/void morphology. The area available for surface modification is enhanced by smaller pores, while molecular transport benefits from the network of larger pores. The bimodal architecture, which is the result of a series of fabrication steps, is visualized using scanning electron microscopy (SEM) as a network of pores that are less than 100 nm in size and connected by ligaments to larger pores that are several hundred nanometers in size. The electrochemically active surface area of the hb-NPG is assessed using cyclic voltammetry (CV), with a focus on the critical roles that both dealloying and annealing play in creating the necessary structure. The adsorption of different proteins is measured by solution depletion technique, revealing the better performance of hb-NPG in terms of protein loading. By changing the surface area to volume ratio, the created hb-NPG electrode offers tremendous potential for biosensor development. The manuscript discusses a scalable method to create hb-NPG surface structures, as they offer a large surface area for the immobilization of small molecules and improved transport pathways for faster reactions.

Facile fabrication of hierarchically nanostructured gold electrode for bio-electrochemical applications.

Nanoporous gold (NPG) is one of the most extensively investigated nanomaterials owing to its tunable pore size, ease of surface modification, and range of applications from catalysis, actuation, and molecular release to the development of electrochemical sensors. In an effort to improve the usefulness of NPG, a simple and robust method for the fabrication of hierarchical and bimodal nanoporous gold electrodes (hb-NPG) containing both macro-and mesopores is reported using electrochemical alloying and dealloying processes to engineer a bicontinuous solid/void morphology. Scanning electron microscopy (color SEM) images depict the hierarchical pore structure created after the multistep synthesis with an ensemble of tiny pores below 100 nm in size located in ligaments spanning larger pores of several hundred nanometers. Smaller-sized pores are exploited for surface modification, and the network of larger pores aids in molecular transport. Cyclic voltammetry (CV) was used to compare the electrochemically active surface area of the hierarchical bimodal structure with that of the regular unimodal NPG with an emphasis on the critical role of both dealloying and annealing in creating the desired structure. The adsorption of different proteins was followed using UV-vis absorbance measurements of solution depletion revealing the high loading capacity of hb-NPG. The surface coverage of lipoic acid on the hb-NPG was analyzed using thermogravimetric analysis (TGA) and reductive desorption. The roughness factor determinations suggest that the fabricated hb-NPG electrode has tremendous potential for biosensor development by changing the scaling relations between volume and surface area which may lead to improved analytical performance. We have chosen to take advantage of the surface architectures of hb-NPG due to the presence of a large specific surface area for functionalization and rapid transport pathways for faster response. It is shown that the hb-NPG electrode has a higher sensitivity for the amperometric detection of glucose than does an NPG electrode of the same geometric surface area.

Nanomaterials for Biosensing Lipopolysaccharide.

Lipopolysaccharides (LPS) are endotoxins, hazardous and toxic inflammatory stimulators released from the outer membrane of Gram-negative bacteria, and are the major cause of septic shock giving rise to millions of fatal illnesses worldwide. There is an urgent need to identify and detect these molecules selectively and rapidly. Pathogen detection has been done by traditional as well as biosensor-based methods. Nanomaterial based biosensors can assist in achieving these goals and have tremendous potential. The biosensing techniques developed are low-cost, easy to operate, and give a fast response. Due to extremely small size, large surface area, and scope for surface modification, nanomaterials have been used to target various biomolecules, including LPS. The sensing mechanism can be quite complex and involves the transformation of chemical interactions into amplified physical signals. Many different sorts of nanomaterials such as metal nanomaterials, magnetic nanomaterials, quantum dots, and others have been used for biosensing of LPS and have shown attractive results. This review considers the recent developments in the application of nanomaterials in sensing of LPS with emphasis given mainly to electrochemical and optical sensing.