Early diagnosis of acute kidney injury by aptasensors

 描述（由申请人提供）：急性肾损伤（阿基）每年影响全球超过1000万人，不同患者人群的报告死亡率为15%至60%。然而，缺乏方法来支持阿基的早期诊断，这可能导致早期干预，改善治疗，改善预后和降低医疗成本。由于这些原因，开发阿基的早期诊断工具是NIDDK列出的征集研究主题之一。中性粒细胞明胶酶相关脂质运载蛋白（NGAL）是阿基的可靠生物标志物，也可能具有一些预测用途。 我们建议开发一种便携式设备，用于在护理点进行可靠的阿基早期检测。我们的提案的创新在于将适体技术与NGAL的微悬臂检测相结合，NGAL是阿基的早期标志物[阿基发作时2- 4小时;截止值约为150 ng/ml]。适体是单链DNA或RNA，其与其靶物强烈且特异性地结合，并且通过指数富集配体系统进化（SELEX）在体外选择。对于生物传感器的开发，适体相对于抗体具有许多优势，包括尺寸较小、易于化学合成和修饰以及适用于包括微悬臂检测系统在内的广泛测定形式。微悬臂梁是一种通常由硅制成的梁，厚度为1-5微米，长度为500-100微米，仅在一端固定。当微悬臂梁的表面应力由于其表面上的配体-受体（例如适体-靶标）相互作用而发生构象变化而改变时，微悬臂梁弯曲。通过干涉测量法测量涂覆有不结合分析物的乱序核酸的参考悬臂和涂覆有结合分析物的适体的感测悬臂之间的表面应力差。 在第一阶段的研究中，我们分离出几个适体，结合NGAL和证明的能力，其中之一，以感NGAL时，适体连接到微悬臂梁。在第二阶段研究中，我们提出以下具体目标：1）比较几种适体的特异性、在血清蛋白存在下的功能性、对NGAL的亲和力、在血浆和尿的盐混合物特征存在下的有效性以及与微悬臂装置的相容性，2）优化表现最好的适体的长度、序列、稳定性以及在血液和尿中的最佳测定性能，以及3）优化用于适体传感的微悬臂梁装置（用于检测的1-100 nM的动态范围，信噪比> 10，协方差< 10%）。将微悬臂梁装置小型化到等于或小于鞋盒的尺寸。 从这些研究中，我们希望开发一种优化的和可靠的适配体功能化的微悬臂梁，通过它可以定量NGAL在血浆和尿液样品中具有强大的信号/噪声比，灵敏度，检测限和测量的动态范围。III期研究将涉及制造和验证用于监测NGAL的设备，以在护理点检测阿基。

 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.