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

项目摘要阿尔茨海默病(AD)是一个主要的医疗保健和社会经济负担。使用 有效的治疗方法尚待开发，在前驱阶段及早发现和监测 进展是控制和干预AD发展的最好方法。而非侵入性正电子 发射断层扫描可以测量大脑中的淀粉样多肽(AββS)和微管tau蛋白 确认AD的发病、成像设备的可用性、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曝光限制了早期筛查的成像应用 在监测疾病进展方面，大量和多样化的人口和重复扫描。因此，有一个 在开发强大、可获得和成本效益高的体外诊断工具方面的需求尚未得到满足 高危患者脑脊液甚至血液中A、β、S和tau蛋白水平的检测 在“看护点”的个人。然而，这些AD生物标志物的血清检测需要高灵敏度 由于A、β、S或tau蛋白在血液样本中浓度较低，且变化缓慢且逐渐 这些生物标志物在疾病进展过程中的水平。当前以生物胺为基础的一大挑战 在体外诊断是生物污垢效应，即蛋白质和生物分子在细胞表面的吸附 检测试剂和设备。这会导致不需要的分子层的形成，从而减少 通过(1)阻断针对目标生物标志物的抗体，以及(2)引起 来自吸附的非特异性蛋白质和其他分子的高“背景噪声”干扰检测 靶向生物标记物。我们相信，这些问题可以通过我们的创新解决方案来克服，即：1) 抗生物污垢聚合物包覆纳米粒子捕捉剂，以阻断非特异性蛋白质的吸附，从而 保护靶向配体/抗体的敏感性和特异性；2)高磁性磁铁 具有纳米磁性搅拌棒作用的氧化物纳米棒(IONRs)，以增强捕捉剂和 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 一种具有超高灵敏度和特异度的基于多路复用的血清“液体活检”系统的研制 AD生物标志物Aβ40、Aβ42、总Tau、磷酸化Tau和淀粉样蛋白的检测 一个样本中的前体蛋白(APP)。我们将制作和优化抗体偶联的抗生物污垢IONRs 具有不同长宽比的高效流体混合和添加Aβ40、Aβ42、TTau的多路检测 以及血液样本中的ptau蛋白和APP669-711(目标1)，同时开发一种具有 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 通过多路检测血清Aβ40、Aβ42、tTau、ptau蛋白和 血清APP669-711水平与血清APP669-711水平 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(目标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).