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

项目摘要 囊性纤维化（CF）是一种使人衰弱的进行性肺部疾病，目前尚无治愈方法。通过适当 管理，患者可以获得良好的症状控制和生活质量。急性加重为急性发作 呼吸道症状恶化，通常由感染引发。急性加重期间，中性粒细胞过多 弹性蛋白酶（NE）被释放到肺中并对肺组织造成不可逆的损伤。这种酶可以 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 肺组织损伤因此，目的是开发一种便携式的即时检测方法， CF患者监测痰液中NE活性。 该测试平台将包括一个磁性芯片阅读器和一个一次性生物传感器芯片， 用连接到磁性纳米颗粒（MNP）的NE特异性可切割肽功能化。肽将 用作传感器表面和MNP之间的连接物。NE切割肽将释放MNP 导致信号随时间定量下降。肽裂解速率与NE相关 丰饶。因此，当痰中NE增加时，该设备将通知临床医生急性加重 开始了 检测和定量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 用于肽的最佳附着的化学物质。为了促进NE的有效切割，我们将优化 肽底物的密度和长度。优化后，肽底物将固定在 磁性传感器表面和96孔板中使用的荧光报告分子将被MNP替换。在 目的2，加入纯化的NE或患者痰样品将导致肽的切割和 随着MNP从传感器表面释放，磁阻降低。这种免洗蛋白酶 可以实时监测分析，从而实现可以由 未经训练的专业人士。虽然我们提出的研究将集中在CF患者的痰液，但这种测定将是可行的。 适用于患有其他慢性气道疾病的患者，包括慢性阻塞性肺疾病 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).