Rapid Analytics for Endotoxin using Liquid Crystalline Droplets

使用液晶液滴快速分析内毒素

• 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
• 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

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）：在广泛的与呼吸健康相关的环境中，革兰氏阴性细菌内毒素内毒素（脂多糖，LPS）的检测和量化至关重要很重要机载内毒素）和（iii）设备和试剂安全性（基于气雾剂的治疗剂以及临床呼吸器设备的不育验证）。在这些不同的背景下，Limulus Amoebocyte裂解物（LAL）分析被广泛使用。但是，在许多方面，内毒素检测的LAL分析并不是最佳 - 它是复杂的，它采用了从马蹄蟹大杂环中提取的不稳定的酶混合物：LAL酶混合物的活性的复杂性和变化，这是由于许多干扰化合物以及crab crab sectss semits semits secipits semite secipits secipits secipits secipits secipits semi -sem -sem -sem -sem -sem -sem -semi -sem -sem -sem -sem -sem -sem -emi -emi -secipits secipity secipity secipits secipits。本文中，我们提出了一种基于使用微米大小的液晶（LCS）的新型，高度敏感和无酶的分析方法，用于光学检测内毒素。这种方法不采用动物来源的试剂，它是便宜的，它是根据最近发现的发现，LP与LC液滴的界面结合可以触发LC在液滴内的订购订购的变化，从而触发其光学外观。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LP，这比商业LAL分析更敏感和更快。 LC液滴的响应是针对LP的，相对于DLPC和DOPC等磷脂以及一系列合成表面活性剂的响应。最后，对LC液滴的光学外观进行定量提供了定量LPS浓度的方法。这些观察结果合并后，表明LC液滴可以提供简单而低成本的基础，但快速，敏感和选择性的方法可以在各种医疗和工业环境中获得的水性样品报告LPS，这对于预防和管理气道疾病很重要。该R21赠款的应用程序旨在为工程师，生命科学家，基础和临床医学研究人员组成的跨学科团队核定，以解决可以评估这些最新发现的关键问题。为此，AIM 1将严格定义LC液滴的分析特性，这是对内毒素定量的广泛有用方法的基础。具体而言，使用来自多种生物医学相关细菌的LPS，我们将确定分析方法的灵敏度，动态范围，特异性和速度。 AIM 2研究将提供对LP和LC液滴之间相互作用机制的基本见解，从而为未来的优化分析方法提供了严格的技术基础：提出了特定的假设，以测试脂质A在LCS订购订单中的结构的作用。 公共卫生相关性：此R21应用的重点是开发一种新型的分析方法，用于检测和定量革兰氏阴性细菌内毒素。分析方法不需要不稳定的生物试剂，低成本，并且特别适合低资源环境。该技术有可能在各种与呼吸健康相关的环境中影响内毒素的检测和定量，包括临床和基本生物医学研究，公共和职业健康以及设备和药物的安全。