A microchip to analyze trafficking leukocytes in Alzheimer’s disease patients

 描述(申请人提供)：阿尔茨海默病(AD)是最常见的神经退行性疾病之一，会导致进行性记忆障碍和认知障碍等有害后果。尽管预计AD的患病率将在未来30年内翻一番，但目前还没有被广泛接受的分子生物标志物用于AD的早期检测或非侵入性监测。越来越多的证据表明，免疫反应和脑部炎症参与了中枢神经系统退行性疾病的发病机制。参与脑深部炎症反应的免疫细胞往往进入脑脊液，即中枢神经系统的循环系统，这些细胞携带着有关脑深部炎症病理的信息。另一方面，血脑屏障限制了免疫细胞进入中枢神经系统，但在病理生理状态下，少量免疫细胞可以进入中枢神经系统参与免疫监视。这些免疫细胞的过度迁移或功能异常会导致神经退行性病变的发生。这些在脑脊液中运输的白细胞被认为是检测和测量炎性神经退行性疾病的潜在细胞标志物。然而，这仍然具有挑战性，因为(1)运输白细胞(~1个/微升)和(2)细胞高度异质性，具有不同的免疫效应功能/细胞分泌的蛋白质(高达40)。Isoplexis拥有一项手持原型技术，首次提供了在单细胞水平上测量许多(多达45个)这些关键效应蛋白的能力。同时，这种设想形式的设备需要更少的细胞输入量(~1000)，与现有的单细胞仪器(如流式细胞仪)相比，这是一个重大优势，用于分析稀有贩运的白细胞。它的成本也将比现有的单细胞仪器低得多，这代表着显著的市场优势。因此，我们计划开发一个完全集成的系统，包括利用纳米颗粒表面浓缩贩运白细胞和在同一微型设备上进行单细胞效应蛋白分析，以真正使贩运白细胞真正成为广泛使用的机会，作为炎症性神经退行性疾病(特别是AD)的早期诊断和监测的生物标志物。为了达到这一目标，我们建议：1.在Isoplexis微设备中加入细胞捕获模块，执行低丰度白细胞的芯片分离，然后进行高度多元化的免疫功能分析。2.研制了一种集成载体装置，实现了检测脑脊液稀有白细胞的集成芯片的可靠运行。我们期望开发一种独特的微创方法来定量测量大脑深部的炎症情况，以便使用脑脊液对AD进行早期诊断和治疗监测。这一方法也将对临床前或临床用于炎症性神经退行性疾病常规筛查或监测的应用产生广泛影响。

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is one of the most common neurodegenerative diseases that lead to detrimental outcomes such as progressive memory deficit and cognitive impairment. Although it is expected that the prevalence of AD will double over the next 30 years, currently no widely-accepted molecular biomarkers for early detection or non-invasive monitoring of AD. There has been increasing evidence that immune responses and brain inflammation are involved in the pathogenesis of neurodegenerative disorders in the central nervous system (CNS). The immune cells participating in the inflammatory response in the deep brain often get into cerebrospinal fluid (CSF), so called the "circulatory" system of CNS, and these cells carry the information about deep brain inflammatory pathology. On the other hand, while the blood-brain barrier (BBB) restricts the entry of immune cells into the CNS, a small number of immune cells can traverse into the CNS during pathophysiological states to participate in immune surveillance. Excessive migration or abnormal functioning of these immune cells contribute to the development of neurodegenerative pathology. It has been hypothesized that these trafficking leukocytes in CSF are potential cell markers to detect and measure inflammatory neurodegenerative disease. However, it remains challenging due to (i) the paucity of trafficking leukocytes (~1 cell/microliter) and (2) the high degree of cellular heterogeneity with diverse immune effector functions/proteins secreted by cells (up to 40). IsoPlexis has a prototype hand-held technology that for the first time provides the ability to measure many (up to 45) of these key effector proteins at the single cell level. At the same time, this device in its envisioned form requires much less amount of cell input (~1000) representing a major advantage over the existing single-cell instruments (e.g., flow cytometer) for the specific application toward the analysis of rare trafficking leukocytes. It will also be far less costly tha existing single-cell instruments, representing a significant market advantage. Thus, we plans to develop a fully integrated system that incorporates the enrichment of trafficking leukocytes using nanorough surfaces and single-cell effector protein analysis on the same microdevice to truly enable the opportunity for wide-spread use of trafficking leukocytes as the biomarker for early stage diagnosis and monitoring of inflammatory neurodegenerative diseases (specifically AD). To reach this goal, we propose: 1. Incorporating a cell capture module in the IsoPlexis microdevice to perform on-chip separation of low abundance leukocytes followed by highly multiplexed immune function analysis. 2. Develop an integrated carrier device to perform reliable operation of the integrated microchip for measuring rare trafficking leukocytes from CSF. We expect to develop a unique and minimally invasive approach to quantitatively measure inflammatory conditions in deep brain for early diagnosis and therapeutic monitoring of AD using CSF. This approach will also have broad impact on preclinical or clinical uses for routine screening or monitoring of inflammatory neurodegenerative diseases.