Point-of-Care Quantitation of Sputum Neutrophil Elastase Activity in Chronic Airway Disease

慢性气道疾病痰中性粒细胞弹性蛋白酶活性的护理点定量

• 批准号: 9809963
• 负责人: Drew Alexander Hall
• 金额: $ 23.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9809963
• 关键词: Acute; Adult; Airway Disease; Biological Assay; Biological Markers; Biosensor; Biotin; Chemistry; Chronic; Chronic Obstructive Airway Disease; Cleaved cell; Clinic; Cystic Fibrosis; Cystic Fibrosis sputum; Data; Development; Devices; Digestion; Enzymes; Event; Home environment; Immobilization; Infection; Laboratories; Lead; Length; Leukocyte Elastase; Longitudinal Studies; Lung; Lung diseases; Magnetic nanoparticles; Magnetism; Modification; Monitor; Patients; Peptide Hydrolases; Peptides; Preparation; Procedures; Quality of life; Reader; Recombinants; Reporter; Research; Resistance; Respiratory Signs and Symptoms; Running; Sampling; Signal Transduction; Sputum; Streptavidin; Structure of parenchyma of lung; Surface; Symptoms; Testing; Time; Translating; cystic fibrosis patients; density; effective therapy; experience; instrumentation; lung injury; nanosensors; neutrophil; point of care; portability; recruit; sensor

PROJECT SUMMARY Cystic fibrosis (CF) is a debilitating, progressive lung disease that currently has no cure. With proper management, patients can achieve good symptom control and quality of life. Exacerbations are acute episodes of worsening of respiratory symptoms, often triggered by an infection. During exacerbations, excess neutrophil elastase (NE) is released into the lung and causes irreversible damage to lung tissue. This enzyme can be detected in sputum and therefore serves as a biomarker for acute exacerbations. We hypothesize that daily, remote monitoring of this enzyme in CF patient sputum would result in more effective treatment and a reduction in lung tissue damage. Therefore, the objective is to develop a portable, point-of-care assay that can be used by CF patients to monitor NE activity in their sputum. The testing platform will consist of a magnetic chip reader and a disposable biosensor chip that has been pre- functionalized with NE-specific cleavable peptides attached to magnetic nanoparticles (MNPs). The peptides will serve as linkers between the sensor surface and the MNPs. Cleavage of peptides by NE will release the MNP resulting in a quantitative decrease in signal over time. The rate of peptide cleavage correlates with NE abundance. Therefore, when NE increases in sputum, this device will notify clinicians that an acute exacerbation is beginning. The ability to detect and quantify NE activity will be determined by how well this enzyme can cleave surface immobilized peptide substrates. Therefore, in Aim 1, we will use 96-well plate assays to evaluate different surface chemistries for optimal attachment of the peptide. To facilitate efficient cleavage by NE, we will optimize the density and length of the peptide substrate. Once optimized, the peptide substrate will be immobilized to the magnetic sensor surface and the fluorescent reporter used in the 96-well plates will be replaced with MNPs. In Aim 2, addition of either purified NE or patient sputum samples will result in cleavage of the peptide and a decrease in magnetic resistance as the MNPs are released from the sensor surface. This wash-free protease assay can be monitored in real-time enabling a “sample-to-answer” testing procedure that can be run by untrained professionals. While our proposed research will focus on sputum from CF patients, this assay will be suitable for use by patients with other chromic airway diseases, including Chronic Obstructive Pulmonary Disease (COPD).

项目概要 囊性纤维化 (CF) 是一种使人衰弱的进行性肺部疾病，目前无法治愈。与适当的 通过管理，患者可以获得良好的症状控制和生活质量。病情加重是急性发作 呼吸道症状恶化，通常由感染引起。病情加重期间，中性粒细胞过多 弹性蛋白酶（NE）被释放到肺部并对肺组织造成不可逆的损伤。这种酶可以是 在痰液中检测到，因此可作为急性加重的生物标志物。我们假设每天， 远程监测 CF 患者痰中的这种酶将导致更有效的治疗并减少 在肺组织损伤中。因此，我们的目标是开发一种便携式即时检测方法，可供 CF 患者监测痰中的 NE 活性。 该测试平台将由一个磁性芯片读取器和一个预先准备好的一次性生物传感器芯片组成。 通过附着在磁性纳米颗粒 (MNP) 上的 NE 特异性可裂解肽进行功能化。这些肽将 作为传感器表面和 MNP 之间的连接器。 NE 裂解肽会释放 MNP 导致信号随着时间的推移而减少。肽裂解率与 NE 相关 丰富。因此，当痰液中 NE 增加时，该装置会通知临床医生病情急性加重 正在开始。 检测和量化 NE 活性的能力将取决于该酶切割表面的能力 固定化肽底物。因此，在目标 1 中，我们将使用 96 孔板测定来评估不同的表面 用于肽最佳附着的化学物质。为了促进 NE 的有效裂解，我们将优化 肽底物的密度和长度。一旦优化，肽底物将被固定到 96 孔板中使用的磁传感器表面和荧光报告器将被 MNP 取代。在 目标 2，添加纯化的 NE 或患者痰样本将导致肽的裂解和 当 MNP 从传感器表面释放时，磁阻降低。这种免洗蛋白酶 可以实时监控测定，从而实现“样本到答案”测试程序，该程序可以通过以下方式运行 未经培训的专业人员。虽然我们提出的研究将重点关注 CF 患者的痰液，但该测定将 适合患有其他慢性气道疾病（包括慢性阻塞性肺疾病）的患者使用 疾病（慢性阻塞性肺病）。