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).

项目摘要 囊性纤维化（CF）是一种令人衰弱的，进行性肺部疾病，目前尚无治愈。适当 管理，患者可以实现良好的症状控制和生活质量。加剧是急性情节 担心呼吸道症状，通常是由感染引起的。在加剧期间，超过中性粒细胞 弹性酶（NE）被释放到肺部，并对肺组织造成不可逆转的损害。这个酶可以是 在痰液中检测到，因此是急性加重的生物标志物。我们每天假设这一点， 在CF患者痰液中远程监测该酶会导致更有效的治疗和减少 在肺组织损伤中。因此，目的是开发一种便携式的，即保健的测定法 CF患者监测痰液中的NE活性。 测试平台将由磁芯片读取器和一次性生物传感器芯片组成 用连接到磁性纳米颗粒（MNP）的NE特异性切解辣椒官能化。辣椒会 用作传感器表面和MNP之间的接头。 NE裂解Petides将释放MNP 随着时间的流逝，信号的定量减少。胡椒裂解的速率与NE相关 丰富。因此，当NE增加痰液时，该设备将通知临床医生急性加重 正在开始。 检测和量化NE活性的能力将取决于该酶清除表面的能力 固定的肽底物。因此，在AIM 1中，我们将使用96孔板测定法来评估不同的表面 化学以最佳的肽附着。为了促进NE的有效分裂，我们将优化 肽底物的密度和长度。一旦优化，肽底物将被固定在 磁传感器表面和96孔板中使用的荧光记者将被MNP替换。在 AIM 2，添加纯化的NE或患者痰液样品将导致胡椒和A裂解 当MNP从传感器表面释放时，磁电阻的降低。这种无洗的蛋白酶 可以实时监控测定，启用可以通过 未经训练的专业人员。虽然我们提出的研究将侧重于CF患者的痰液，但此测定将是 适用于其他铬酸气道疾病的患者使用，包括慢性阻塞性肺 疾病（COPD）。