The detection and quantification of Gram-negative bacteria endotoxin (lipopolysaccharide, LPS) is critically important in a wide range of respiratory health-related contexts, including (i) clinical and basic biomedical research (nosocomial infection, including airway infections which are a major cause of mortality in hospital intensive care units), (ii) public and occupational health (measurement of airborne endotoxin), and (iii) device and agent safety (aerosol-based therapeutic agents and validation of sterility of clinical respiratory devices). In these diverse contexts, the Limulus amoebocyte lysate (LAL) assay is widely employed. However, in many respects, the LAL assay for endotoxin detection is not optimal - it is complex, employing a labile mixture of enzymes extracted from horseshoe crab amoebocytes: The complexity and variation in the activity of the LAL enzyme mixtures, which results from numerous interfering compounds as well as variation amongst crab species, leads to semi-quantitative results. Herein, we propose a novel, highly sensitive and enzyme-free analytical methodology for optical detection of endotoxin that is based on the use micrometer-sized droplets of liquid crystals (LCs). This approach does not employ animal-derived reagents, it is inexpensive, and it builds from the recent discovery that LPS binding to the interfaces of LC droplets can trigger changes in the ordering of LCs within the droplets and thus their optical appearances. Preliminary studies have revealed that the LC- based method, even prior to optimization, can detect LPS with a sensitivity of 0.1-1 pg/mL within one minute, which is more sensitive and faster than commercial LAL assays. The response of the LC droplets is specific to LPS relative to phospholipids such as DLPC and DOPC and a range of synthetic surfactants. Finally, quantification of the optical appearance of the LC droplets offers approaches to quantification of the LPS concentration. These observations, when combined, suggest that LC droplets may offer the basis of a simple and low cost, yet rapid, sensitive and selective method for reporting LPS in aqueous samples obtained in a variety of medical and industrial settings important for the prevention and management of airway disease. This R21 grant application seeks to nucleate an interdisciplinary team of engineers, life scientists, basic and clinical medical researchers to address key questions that will enable assessment of these recent discoveries. To this end, Aim 1 will rigorously define the analytic characteristics of LC droplets as the basis of a broadly useful methodology for the quantification of endotoxin. Specifically, using LPS from multiple strains of biomedically relevant bacteria, we will determine the sensitivity, dynamic range, specificity and speed of the analytic methodology.
Aim 2 research will provide fundamental insights into the mechanisms of interaction between LPS and LC droplets thereby providing a rigorous technical foundation for future optimization of the analytic method: Specific hypotheses are formulated to test the role of the structure of lipid A on the ordering of LCs.
This R21 application is focused on the development of a novel analytical methodology for detection and quantification of Gram-negative bacterial endotoxin. The analytical methodology does not require labile biological reagents, is low cost, and is particularly well-suited to low-resource environments. The technology has the potential to impact the detection and quantification of endotoxin in a wide range of respiratory health-related contexts, including clinical and basic biomedical research, public and occupational health, and device and agent safety.
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