Early diagnosis of acute kidney injury by aptasensors

适体传感器早期诊断急性肾损伤

• 批准号: 8905954
• 负责人: Muslum Ilgu
• 金额: $ 30.96万
• 依托单位: APTALOGIC, INC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905954
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Affect; Affinity; Antibodies; Aptamer Technology; Binding; Biological Assay; Biological Markers; Biosensor; Blood; Blood Proteins; Characteristics; Chemicals; Classification; Clinics and Hospitals; Cost Analysis; Creatinine; Detection; Development; Device or Instrument Development; Devices; Diabetes Mellitus; Diagnosis; Digestive System Disorders; Early Diagnosis; Early Intervention; Early treatment; Effectiveness; Evolution; Funding; Future; Gelatinase A; Goals; Hour; In Vitro; Institutes; Intensive Care Units; Interferometry; Intervention; Kidney Diseases; Label; Laboratories; Lead; Length; Life; Ligands; Measurement; Measures; Medical; Methods; Modification; Molecular Conformation; Monitor; National Institute of Diabetes and Digestive and Kidney Diseases; Noise; Nucleic Acids; Onset of illness; Output; Patient Monitoring; Patients; Performance; Phase; Plasma; Population; Procedures; Process; Proteins; Quantitative Evaluations; RNA; RNA Binding; Renal function; Reporting; Research; Risk; Role; Sales; Sampling; Serum; Serum Proteins; Signal Transduction; Silicon; Single-Stranded DNA; Sodium Chloride; Specificity; Stress; Surface; System; Technology; Temperature; Thick; Time; Urea; Urine; Vision; aptamer; base; cantilever; chemical synthesis; cost; improved; innovation; instrument; miniaturize; monitoring device; mortality; outcome forecast; patient population; phase 1 study; phase 2 study; phase 3 study; point of care; public health relevance; receptor; research and development; response; sensor; tool

 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.

 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，据报道不同患者群体的死亡率为 15% 至 60%。然而，缺乏支持 AKI 早期诊断的方法，从而实现早期干预、改善治疗、更好的预后和更低的医疗成本。因此，AKI早期诊断工具的开发成为NIDDK列出的征集研究课题之一。中性粒细胞明胶酶相关脂质运载蛋白 (NGAL) 是 AKI 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，用于在护理点可靠地早期检测 AKI。我们提案的创新之处在于将适体技术与微悬臂梁检测 NGAL 相结合，NGAL 是 AKI 的早期标志物 [AKI 发病 2-4 小时；截止值~150 ng/ml]。适体是单链 DNA 或 RNA，可与其靶标强烈且特异性地结合，并通过指数富集配体系统进化 (SELEX) 在体外进行选择。对于生物传感器的开发，适配体比抗体具有许多优势，包括尺寸更小、易于化学合成和修饰以及对包括微悬臂梁检测系统在内的多种测定形式的适应性。微悬臂梁是一根梁，通常由硅制成，厚 1-5 µm，长 500-100 µm，仅在一端固定。当其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发生变化时，微悬臂梁会弯曲。通过干涉测量法测量涂有不结合分析物的乱序核酸的参考悬臂和涂有结合分析物的适体的传感悬臂之间的差异表面应力。 在一项 I 期研究中，我们分离了几种结合 NGAL 的适体，并证明了当适体附着在微悬臂梁上时，其中一种能够感知 NGAL。在这项 II 期研究中，我们提出以下具体目标：1) 比较几种适体的特异性、血清蛋白存在下的功能性、NGAL 的亲和力、血浆和尿液特有的盐混合物存在下的有效性以及与微悬臂梁装置的兼容性，2) 优化性能最佳的适体的长度、序列、稳定性和血液和尿液中的最佳检测性能，以及 3) 优化适体的长度、序列、稳定性和血液和尿液中的最佳检测性能。 用于适体传感的微悬臂梁装置（检测动态范围为 1-100 nM，信噪比 > 10，协方差 < 10%）。将微悬臂梁装置小型化至等于或小于鞋盒的尺寸。 通过这些研究，我们期望开发出一种优化且可靠的适配体功能化微悬臂梁，通过该微悬臂梁可以对血浆和尿液样本中的 NGAL 进行定量，并具有稳健的信噪比、灵敏度、检测限和动态测量范围。第三阶段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 AKI。