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

项目总结 囊性纤维化是一种衰弱的进行性肺部疾病，目前尚无治愈方法。有了适当的 通过管理，患者可以实现良好的症状控制和生活质量。急性发作是急性发作。 通常由感染引发的呼吸道症状的恶化。在病情恶化期间，过量的中性粒细胞 弹性蛋白酶(NE)被释放到肺内，对肺组织造成不可逆转的损害。这种酶可以是 在痰中检测到，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和减少 在肺组织损伤方面。因此，我们的目标是开发一种便携的、可供 Cf患者监测其痰中NE的活性。 测试平台将由磁性芯片读取器和已预先安装的一次性生物传感器芯片组成 与磁性纳米颗粒(MNPs)连接的NE特定的可裂解多肽功能化。这些多肽将 作为传感器表面和MNPs之间的连接物。去甲肾上腺素对多肽的裂解将释放MNP 从而导致信号随着时间的推移而定量减少。肽的裂解率与去甲肾上腺素相关 富足。因此，当痰中NE增加时，该设备会通知临床医生急性加重 才刚刚开始。 检测和量化去甲肾上腺素活性的能力将取决于这种酶切割表面的能力 固定化多肽底物。因此，在目标1中，我们将使用96孔板分析来评估不同的表面 优化多肽附着的化学方法。为了促进NE的有效切割，我们将优化 多肽底物的密度和长度。一旦优化，多肽底物将被固定在 96孔板中使用的磁性传感器表面和荧光报告将被MNPs取代。在……里面 目的2，加入纯化的NE或患者痰样本将导致多肽和a的裂解 当MNPs从传感器表面释放时，磁阻降低。这种免洗的蛋白水解酶 可以实时监控化验结果，从而实现可通过以下方式运行的“样本到答案”测试程序 未受过训练的专业人士。虽然我们提议的研究将集中在CF患者的痰上，但这项检测将是 适用于其他慢性呼吸道疾病患者，包括慢性阻塞性肺疾病患者 疾病(COPD)。

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).