Early diagnosis of acute kidney injury by aptasensors

描述（由申请人提供）：急性肾损伤（AKI）每年影响全球超过1000万人，不同患者群体的死亡率从15%到60%不等。然而，缺乏支持AKI早期诊断的方法，从而导致早期干预，改善治疗，改善预后和降低医疗费用。基于这些原因，AKI早期诊断工具的开发是NIDDK列出的征求研究课题之一。我们的最终目标是开发一种便携式设备，在护理点可靠地早期检测AKI。本研究的创新之处在于将适体技术与血清/尿中性粒细胞明胶酶相关脂钙蛋白（NGAL）的微悬体检测相结合，NGAL是AKI的早期标志物[AKI发病2-4小时，截止浓度为~ 150 ng/ml (6.8 nM)]。适配体是通过指数富集（SELEX）系统进化配体（Systematic Evolution of Ligands by EXponential enrichment）在体外筛选出来的能够快速特异性结合靶标的ssdna / rna。对于生物传感器的开发，适配体比抗体有许多优点，包括它们更小的尺寸，易于化学合成和修饰，以及对包括微悬臂检测系统在内的广泛检测格式的适应性。微悬臂是一种通常由硅制成的梁，厚度为1-5米，长度为500-100米，仅在一端固定。由于配体-受体（如适体-靶标）在其表面相互作用时发生构象变化，微悬臂梁的表面应力发生变化，从而发生弯曲。用干涉测量法测量涂有不与分析物结合的混乱核酸的参考悬臂梁和涂有与分析物结合的适体的传感悬臂梁之间的表面应力差。在第一阶段的研究中，我们提出了两个具体目标：1。鉴定具有纳米级亲和力的2' f修饰的适配体特异性结合NGAL。2. 演示用于NGAL检测的适配体功能化微悬臂装置。在第一阶段，我们期望开发一种适配体功能化的微悬臂原型，该原型将对NGAL做出反应，第二阶段将进一步优化其在血清和尿液样本中的可靠性能，具有稳健的信噪比、灵敏度、检测极限和动态测量范围，并可用于护理点监测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. Our ultimate goal is 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 serum/urine neutrophil gelatinase-associated lipocalin (NGAL), an early marker of AKI [2-4 hours at the onset of AKI with cutoff concentration of ~ 150 ng/ml (6.8 nM)]. Aptamers are ssDNAs/RNAs that bind their targets avidly and specifically, and are selected in vitro by Systematic Evolution of 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 ¿m thick and 500-100 ¿m 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 this phase I study, we propose two specific aims: 1. Identify 2'F-modified aptamers that bind NGAL specifically with nanomolar affinity. 2. Demonstrate an aptamer-functionalized microcantilever device for NGAL detection. In phase I, we expect to develop a prototype of an aptamer-functionalized microcantilever that will be responsive to NGAL, which will be further optimized in phase II for reliable performance with serum and urine samples with robust signal/noise ratio, sensitivity, detection limit and dynamic range of measurement and that can be used at point of care to monitor for AKI.