Rapid Analytics for Endotoxin using Liquid Crystalline Droplets

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背景下至关重要，包括(i)临床和基础生物医学研究（医院感染，包括气道感染，这是医院重症监护病房死亡的主要原因），（ii）公共和职业卫生（空气中内毒素的测量），（iii）器械和制剂安全性（气雾剂治疗制剂和临床呼吸器械无菌性验证）。在这些不同的情况下，鲎变形虫细胞裂解（LAL）测定被广泛采用。然而，在许多方面，LAL检测内毒素的方法并不是最佳的——它是复杂的，使用从马蹄蟹变形虫细胞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，这是由许多干扰化合物和螃蟹物种之间的差异造成的，导致半定量结果。在此，我们提出了一种新的、高灵敏度和无酶的内毒素光学检测分析方法，该方法基于使用微米大小的液晶液滴（lc）。该方法不使用动物衍生试剂，价格低廉，并且基于最近的发现，LPS与LC液滴的界面结合可以触发液滴内LC的顺序变化，从而改变其光学外观。初步研究表明，即使在优化之前，基于LC的方法也可以在1分钟内以0.1-1 pg/mL的灵敏度检测LPS，比商业LAL测定法更灵敏，更快。相对于磷脂（如DLPC和DOPC）和一系列合成表面活性剂，LC液滴对LPS的响应是特异性的。最后，LC液滴光学外观的定量提供了定量LPS浓度的方法。这些观察结果综合起来表明，LC液滴可能为在各种医疗和工业环境中获得的水样中报告LPS提供一种简单、低成本、快速、敏感和选择性的方法基础，这对预防和管理气道疾病很重要。这项R21资助申请旨在建立一个由工程师、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，Aim 1将严格定义LC液滴的分析特性，作为内毒素定量的广泛有用方法的基础。具体来说，我们将使用来自多个生物医学相关细菌菌株的LPS，确定分析方法的灵敏度、动态范围、特异性和速度。Aim 2的研究将为LPS与LC液滴之间的相互作用机制提供基本的见解，从而为未来分析方法的优化提供严格的技术基础：制定具体的假设来测试脂质a结构对LC排序的作用。

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.