High-sensitivity Immunomagnetic System for "Liquid Biopsy" of Alzheimer's Disease

阿尔茨海默病（AD）是一种主要的医疗保健和社会经济负担。与 有效的治疗方法尚待开发，在前驱期的早期发现和监测 进展是控制和干预AD发展的最佳途径。虽然非侵入性正电子 发射断层扫描（PET）可以测量大脑中的淀粉样β肽（Aβs）和微管tau蛋白， 确认AD的发病、影像设备的可用性、单个Aβ或tau特异性的诊断准确性 放射性示踪剂和放射性材料的成本和暴露限制了早期筛查的成像应用 在监测疾病进展方面，因此，有一个 在开发用于检测和治疗癌症的强大、可获得和具有成本效益的体外诊断工具方面， 测量患者或高危人群脑脊液甚至血液样本中Aβs和tau蛋白的水平， 个人在“护理点”。然而，这些AD生物标志物的血清检测需要高灵敏度 由于血液样本中Aβs或tau蛋白的浓度较低， 这些生物标志物在疾病进展期间的水平。目前基于生物标本的一个主要挑战是 体外诊断是生物污染效应，即，蛋白质和生物分子在表面上的吸附 检测代理和设备。这导致不需要的分子层的形成，这降低了 通过（1）阻断针对靶生物标志物的抗体，和（2）引起 来自吸附的非特异性蛋白质和其他分子的高“背景噪音”干扰了 目标生物标志物。我们相信，这些问题可以通过我们的创新解决方案来解决，即，1)的 抗生物污染聚合物包覆纳米颗粒捕获剂以阻断非特异性蛋白质吸附， 保护靶向配体/抗体的敏感性和特异性; 2）高磁性磁性铁 氧化物纳米棒（IONR），具有纳米尺寸的磁力搅拌棒作用，以增强捕获剂的混合， 微流控检测系统中的靶向生物标志物。通过整合这些技术，我们的项目旨在 开发一种基于多路复用的“液体活检”系统，对血清具有超高灵敏度和特异性 检测选定的AD生物标志物，Aβ40、Aβ42、总-Tau（tTau）、磷酸化-Tau（pTau）181和淀粉样蛋白 前体蛋白（APP）。我们将制备和优化抗体偶联的抗生物污损离子核反应器 具有不同的纵横比，可高效混合液体并多重检测加标的Aβ40、Aβ42、tTau 以及血液样品中的pTau蛋白和APP 669 -711（目标1），结合开发具有 有效的纳米搅拌棒混合，以提高检测效率（目标2），然后评估和验证 通过多重测量血清Aβ40、Aβ42、tTau、pTau蛋白， AD患者中的APP 669 -711水平，并将血清测量值与 对同一患者进行的Aβ和tau PET成像和蛋白质组学分析的结果（目的3）。

Project Summary Alzheimer’s disease (AD) is a major healthcare and social-economic burden. With effective treatment yet to be developed, the early detection at the prodromal stage and surveillance of the progression are the best approach to control and intervene AD development. While non-invasive positron emission tomography (PET) can measure amyloid-β peptides (Aβs) and microtubule tau proteins in the brain to confirm the onset of AD, the availability of imaging equipment, diagnostic accuracy of single Aβ or tau specific radiotracer and the cost and exposure of radioactive materials limit imaging applications from early screening of large and diverse populations and repeated scans in monitoring disease progression. Therefore, there is an unmet need in developing robust, accessible and cost-effective in vitro diagnostic tools for detecting and measuring the levels of Aβs and tau proteins in cerebrospinal fluid or even blood samples of patients or at-risk individuals at the “point-of-care”. However, serum detection of these AD biomarker demands high sensitivity because of low concentrations of Aβs or tau proteins in blood samples and slow and gradual change of the levels of these biomarkers during disease progression. One major challenge to the current biospecimen based in vitro diagnostics is the biofouling effect, i.e., adsorption of proteins and biomolecules on the surface of the detecting agents and devices. This leads to the formation of the layer of unwanted molecules, which reduces the detection specificity and sensitivity by (1) blocking antibodies to the targeted biomarkers, and (2) causing high “background noise” from adsorbed non-specific proteins and other molecules interfering the detection of targeted biomarkers. We believe that these problems can be overcome by our innovative solutions, i.e., 1) the anti-biofouling polymer to coat nanoparticle capturing agents to block the non-specific protein adsorption, thus protecting the sensitivity and specificity of the targeting ligands/antibodies; 2) high magnetism magnetic iron oxide nanorods (IONRs) with nano-sized magnetic stir bar action to enhance mixing of capturing agents and targeted biomarkers in the microfluidic detection system. By integrating these technologies, our project aims to develop a multiplexing based “liquid biopsy” system with ultra-high sensitivity and specificity for serum detection of selected AD biomarkers, Aβ40, Aβ42, total-tau (tTau), phosphorylated-Tau (pTau)181 and amyloid precursor protein (APP) in one sample. We will make and optimize antibody-conjugated anti-biofouling IONRs with different aspect ratios for highly efficient fluid mixing and multiplex detection of spiked Aβ40, Aβ42, tTau and pTau proteins and APP669-711 in blood samples (Aim 1), coupled with developing a microfluidic device with effective nano-stir bar mixing for improved detection efficiency (Aim 2), and then evaluate and validate the developed “liquid biopsy” system by multiplexed measuring serum Aβ40, Aβ42, tTau, pTau proteins and APP669-711 levels in AD patients longitudinally, and comparing and correlating the serum measurement with results from Aβ and tau PET imaging and proteomics analysis done on the same patients (Aim 3).