Rapid Analytics for Endotoxin using Liquid Crystalline Droplets

描述（申请人提供）：革兰阴性菌内毒素的检测与定量脂多糖（lipopolysaccharide，LPS）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，其是医院重症监护病房中死亡的主要原因），（ii）公共和职业健康（测量空气中的内毒素），以及（iii）装置和药剂安全（基于气溶胶的治疗剂和临床呼吸装置的无菌性验证）。在这些不同的情况下，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测定不是最佳的-它是复杂的，采用从鲎变形细胞提取的酶的不稳定混合物：LAL酶混合物的活性的复杂性和变化，其由许多干扰化合物以及蟹种之间的变化引起，导致半定量结果。在此，我们提出了一种新的，高灵敏度和无酶的分析方法，用于光学检测内毒素，这是基于使用微米大小的液滴的液晶（LC）。这种方法不使用动物来源的试剂，它是廉价的，它建立在最近的发现，LPS结合到LC液滴的界面可以触发液滴内LC的排序的变化，从而它们的光学外观。初步研究表明，即使在优化之前，基于LC的方法也可以在一分钟内以0.1-1 pg/mL的灵敏度检测LPS，这比商业LAL测定法更灵敏和更快。相对于磷脂如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液滴的光学外观的量化提供了量化LPS浓度的方法。这些观察结果，当组合时，表明LC液滴可以提供一种简单和低成本的基础，但快速，灵敏和选择性的方法，用于报告在各种医疗和工业环境中获得的水性样品中的LPS，这对预防和管理气道疾病是重要的。这项R21资助申请旨在培养一个由工程师、生命科学家、基础和临床医学研究人员组成的跨学科团队，以解决能够评估这些最新发现的关键问题。为此，目标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。具体而言，使用来自多个生物医学相关细菌菌株的LPS，我们将确定分析方法的灵敏度、动态范围、特异性和速度。目的2研究将提供LPS和LC液滴之间相互作用机制的基本见解，从而为分析方法的未来优化提供严格的技术基础：制定特定假设以测试脂质A结构对LC排序的作用。 公共卫生相关性：该R21申请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，特别适合于低资源环境。该技术有可能在广泛的呼吸系统健康相关环境中影响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器械和试剂安全性。

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.