Rapid Analytics for Endotoxin using Liquid Crystalline Droplets

使用液晶液滴快速分析内毒素

基本信息

批准号：
8197797
负责人：
NICHOLAS L ABBOTT
金额：
$ 18.17万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197797
关键词：
Address Aerosols Animals Appearance Applications Grants Bacteria Benchmarking Binding Biological Biological Assay Biomedical Research Build-it Characteristics Chemical Surfactants Clinical Clinical Research Complex Crabs Detection Development Devices Diagnostic Disease Endotoxins Engineering Environment Enzymes Escherichia Escherichia coli Foundations Future Gram-Negative Bacteria Harvest Health Hospitals Infection Intensive Care Units Laboratories Life Limulus Limulus polyphemus Lipid A Lipopolysaccharides Liquid substance Measurement Medical Methodology Methods Molecular Nosocomial Infections Occupational Health Optics Performance Phagocytes Phospholipids Prevention Pseudomonas Public Health Reagent Relative (related person)Reporting Research Research Personnel Resources Role Safety Salmonella Sampling Scientist Seasons Specificity Speed Sterility Structure Technology Testing Therapeutic Agents Universities Validation Variant Wisconsin Xiphosura aqueous base cost endotoxin binding protein insight liquid crystal mortality novel organizational structure research study respiratory response

项目摘要

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.

描述（由申请人提供）：革兰氏阴性菌内毒素（脂多糖，LPS）的检测和定量在广泛的呼吸系统健康相关领域中至关重要，包括（i）临床和基础生物医学研究（医院感染，包括气道感染是医院重症监护病房死亡的主要原因），(ii) 公共和职业健康（空气内毒素的测量），以及 (iii) 设备和药剂安全（气溶胶治疗）剂和临床呼吸装置的无菌验证）。在这些不同的背景下，鲎变形细胞裂解物 (LAL) 测定被广泛采用。然而，在许多方面，用于内毒素检测的 LAL 测定法并不是最佳的 - 它很复杂，采用从鲎变形细胞中提取的不稳定的酶混合物：LAL 酶混合物活性的复杂性和变化，这是由众多干扰因素造成的化合物以及螃蟹种类之间的差异导致半定量结果。在此，我们提出了一种新颖、高灵敏度、无酶的内毒素光学检测分析方法，该方法基于使用微米级液晶 (LC) 液滴。这种方法不使用动物源性试剂，价格便宜，并且基于最近的发现，即 LPS 与 LC 液滴界面的结合可以触发液滴内 LC 的排序及其光学外观的变化。初步研究表明，基于 LC 的方法，即使在优化之前，也可以在一分钟内以 0.1-1 pg/mL 的灵敏度检测 LPS，这比商业 LAL 检测更灵敏、更快速。相对于磷脂（例如 DLPC 和 DOPC）以及一系列合成表面活性剂，LC 液滴的响应对 LPS 具有特异性。最后，LC 液滴光学外观的量化提供了 LPS 浓度量化的方法。这些观察结果表明，LC 液滴可能为一种简单、低成本、快速、灵敏和选择性的方法提供了基础，用于报告在各种医疗和工业环境中获得的水样中的 LPS，这对于预防和管理非常重要。气道疾病。这项 R21 拨款申请旨在组建一个由工程师、生命科学家、基础和临床医学研究人员组成的跨学科团队，以解决关键问题，从而能够评估这些最新发现。为此，目标 1 将严格定义 LC 液滴的分析特征，作为广泛有用的内毒素定量方法的基础。具体来说，我们将使用来自生物医学相关细菌的多种菌株的 LPS，确定分析方法的灵敏度、动态范围、特异性和速度。目标 2 研究将为 LPS 和 LC 液滴之间的相互作用机制提供基本见解，从而为未来分析方法的优化提供严格的技术基础：制定具体假设来测试脂质 A 结构对 LC 排序的作用。公共健康相关性：该 R21 应用的重点是开发一种用于检测和定量革兰氏阴性细菌内毒素的新型分析方法。该分析方法不需要不稳定的生物试剂，成本低廉，特别适合资源匮乏的环境。该技术有可能影响广泛的呼吸系统健康相关领域内毒素的检测和定量，包括临床和基础生物医学研究、公共和职业健康以及设备和制剂安全。