Early diagnosis of acute kidney injury by aptasensors

 描述(申请人提供)：急性肾损伤(AKI)每年影响全球1000多万人，在不同的患者群体中报告的死亡率从15%到60%。然而，缺乏方法支持AKI的早期诊断，从而导致早期干预、改善治疗、更好的预后和更低的医疗费用。因此，AKI早期诊断工具的开发是NIDDK列出的应邀研究课题之一。中性粒细胞明胶酶相关脂钙蛋白(NGAL)是AKI的可靠生物标志物，也可能具有一定的预测作用。我们建议开发一种便携式设备，用于在护理点可靠地早期检测AKI。我们建议的创新之处在于将适体技术与NGAL的微悬臂式检测相结合，NGAL是AKI的早期标志物[AKI开始时2-4h；截止到~150 ng/ml]。适配子是一种单链DNA或RNA，能与其靶标强烈和特异地结合，并通过指数富集型配体系统进化(SELEX)在体外进行选择。对于生物传感器的开发，适配子比抗体具有许多优势，包括它们更小的尺寸，对化学合成和修饰的适应性，以及对包括微悬臂检测系统在内的多种分析形式的适应性。微悬臂是一种梁，通常由硅制成，厚1-5微米，长500-100微米，只固定在一端。当微悬臂梁表面的应力发生变化时，由于其表面的配体-受体(例如，适体-靶)相互作用而发生构象变化，微悬臂梁发生弯曲。用干涉法测量涂有不结合分析物的杂乱核酸的参比悬臂梁和涂有结合分析物的适配子的传感悬臂梁之间的差异表面应力。在第一阶段的研究中，我们分离了几个与NGAL结合的适配子，并展示了当适配子连接到微悬臂上时，其中一个适配子能够感知NGAL的能力。在这项II期研究中，我们提出了以下具体目标：1)比较几种适配子在血清蛋白存在下的特异性、功能性、对NGAL的亲和力、在血浆和尿液混合盐存在时的有效性以及与微悬臂设备的兼容性；2)从长度、序列、稳定性以及在血液和尿液中的最佳检测性能方面优化性能最佳的适体；3)优化适配子传感的微悬臂装置(检测的动态范围为1-100 nM，信噪比&gt；10，协方差&lt；10%)。将微悬臂装置小型化到等于或小于鞋盒的尺寸。通过这些研究，我们期望开发一种优化和可靠的适体功能化微悬臂梁，用于血浆和尿样中NGAL的定量，具有良好的信噪比、灵敏度、检出限和动态测量范围。第三阶段的研究将包括制造和验证用于监测NGAL的设备，以在护理点检测AKI。

 DESCRIPTION (provided by applicant): Acute kidney injury (AKI) affects more than 10 million people worldwide each year with reported mortalities from 15 to 60% in different patient populations. However, methods are lacking to support the early diagnosis of AKI that could lead to early intervention, improved therapy, better prognosis, and lower medical costs. For these reasons, the development of early diagnosis tools for AKI is one of the solicited research topics listed by the NIDDK. Neutrophil gelatinase-associated lipocalin (NGAL) is a reliable biomarker for AKI and may also have some predictive uses. We propose to develop a portable device for reliable early detection of AKI at point-of-care. The innovation of our proposal lies in the combination of aptamer technology with microcantilever detection of NGAL, an early marker of AKI [2-4h at the onset of AKI; cutoff ~150 ng/ml]. Aptamers are single stranded DNAs or RNAs that bind their targets avidly and specifically, and are selected in vitro by Systematic Evolution f Ligands by Exponential enrichment (SELEX). For biosensor development, aptamers have many advantages over antibodies, including their smaller size, amenability to chemical synthesis and modifications and adaptability to a broad range of assay formats including a microcantilever detection system. A microcantilever is a beam, often of silicon, that is 1-5 µm thick and 500-100 µm long and anchored only at one end. The microcantilever bends when its surface stress changes due to the conformation change that occurs upon ligand-receptor (e.g. aptamer-target) interaction on its surface. The differential surface stress between a reference cantilever coated with a scrambled nucleic acid that does not bind the analyte and a sensing cantilever coated with an aptamer that binds the analyte is measured by interferometry. In a phase I study, we isolated several aptamers that bind NGAL and demonstrated the abilities of one of these to sense NGAL when the aptamer is attached to a microcantilever. In this phase II study, we propose the following specific aims: 1) Compare several aptamers for specificity, functionality in the presence of serum proteins, affinity for NGAL, effectiveness in the presence of salt mixtures characteristic of plasma and urine, and compatibility with the microcantilever device, 2) Optimize the best performing aptamers for length, sequence, stability and optimal assay performance in blood and urine, and 3) Optimize the microcantilever device for aptamer sensing (dynamic range of 1-100 nM for detection, signal/noise ratio > 10, covariance < 10%). Miniaturize the microcantilever device to a size equal to or smaller than a shoebox. From these studies, we expect to develop an optimized and reliable aptamer-functionalized microcantilever by which NGAL can be quantified in plasma and urine samples with robust signal/noise ratio, sensitivity, detection limit and dynamic range of measurement. Phase III studies will involve manufacturing and validating the device for monitoring NGAL to detect AKI at point-of-care.