EFRI- BSBA: Novel Microsystems for Manipulation and Analysis of Immune Cells

EFRI- BSBA：用于免疫细胞操作和分析的新型微系统（Novel Microsystems for Manipulation and Analysis of Immune Cells）项目负责人：Alexander revzin教授领导机构：加州大学戴维斯分校（University of California, Davis）学术价值该提案的目标是开发用于分析和操作免疫细胞的新型微系统。免疫细胞是个体感染、恶性肿瘤和自身免疫性疾病的哨兵。因此，这些细胞可以用来获得诊断信息，以及加强对免疫疾病进展机制的理解。免疫细胞（白细胞）的分析是一个单一的挑战，因为体内存在多种细胞亚群，通常仅根据分泌产物来区分。根据特定蛋白（细胞因子）的分泌，t淋巴细胞可分为辅助性t淋巴细胞1型、辅助性t淋巴细胞2型或辅助性t淋巴细胞17型。类似地，b细胞是一种异质细胞群，仅根据产生的特异性抗体来区分。由于缺乏适当的生物分析工具，目前尚不可能对活t细胞和b细胞分泌的产物进行单细胞水平的实时分析。为了解决这一缺点，本提案将开发新的微系统，允许将免疫细胞排列成高密度的单细胞阵列，然后在单细胞水平上监测分泌蛋白的产生。此外，他们将开发一种“感知和释放”电极阵列系统，用于根据分泌产物识别免疫细胞，随后对这些细胞进行分类/释放。本提案中开发的微系统将用于分析正常和自闭症儿童的t细胞，目的是帮助确定免疫功能和行为异常之间的相关性。单个细胞是构成组织和器官的最小的生命体。因此，在过去的15年里，对单个活细胞的分析一直是生物/生命科学研究的前沿。使用绿色荧光蛋白（GFP）和其他荧光蛋白来报告基因在单个细胞中如何以及何时开启的动态，已经彻底改变了生物和医学科学领域。然而，必须将gfp编码DNA导入细胞的要求限制了报告基因技术在最健壮和最容易转染的细胞中的应用。此外，报告基因技术监测基因表达，而不是蛋白质生产。该提案的愿景是开发一种新的和变革性的生物微系统，用于对难以转染的原代细胞中蛋白质生产的非侵入性动态监测。该提案将侧重于白细胞的分析，并将开发生物传感器和微系统，以监测单个t淋巴细胞的细胞因子产生。跨学科的研究团队将提供基于表面工程和微结构的解决方案，以解决目前困扰单细胞分析的挑战，包括：1)创建高密度单细胞阵列，2)将多分析物传感器与单细胞集成，3)将传感元件与单细胞共定位，以确保分泌代谢物的高局部浓度。在本提案中开发的新型生物传感器将可转化为其他细胞系统（例如循环细胞和锚定依赖细胞），并将广泛应用于生物工程，生物技术和生命科学领域。具有不同专业知识的研究人员在该项目中的跨学科合作为工程和生命科学前沿的中学博士后提供了跨学科教育和培训的独特机会和框架。

ABSTRACT EFRI- BSBA: Novel Microsystems for Manipulation and Analysis of Immune Cells PI: Prof. Alexander RevzinLead Institution: University of California, Davis The Intellectual Merit The goal of the proposal will be the development of novel microsystems for analysis and manipulation of immune cells. Immune cells serve as sentinels of infections, malignancies and autoimmune disorders afflicting an individual. Therefore, these cells may be used to gain diagnostic information as well as to enhance understanding of mechanism of immune disease progression. Analysis of immune cells (leukocytes) represents a singular challenge because there is multiple cell subsets present in the body that are often distinguishable only based on the secreted products. This is true for T-lymphocytes that can be categorized into T-helper 1, T-helper 2 or T-helper 17 phenotype based on the secretion of specific proteins (cytokines). Similarly, B-cells represent a heterogeneous population of cells that are distinguished solely based on the production of specific antibodies. Single cell-level, real-time analysis of products secreted by live T-cells and B-cells is not currently possible due to the lack of appropriate bioanalytical tools. To address this shortcoming, this proposal will develop novel microsystems allowing to arrange immune cells in high density single cell arrays and then monitor production of secreted proteins at a single cell level. In addition, they will develop a "sense-and-release" electrode array system for identifying immune cells based on the secreted product and subsequently sorting/releasing these cells. The microsystems developed in this proposal will be used for the analysis of T-cells from normal and autistic children with the goal of helping to identify correlates between immune function and behavioral aberrations. Broader Impact An individual cell is the smallest living building block of tissues and organs. Therefore, analysis of single living cells has been at the frontier of biological/life science research for the past decade and half. The use of green fluorescence protein (GFP) and other fluorescent proteins to report on dynamics of how and when genes get turned on in individual cells has revolutionized the field of biological and medical sciences. However, the requirement of having to get GFP-encoding DNA into cells limits application of reporter gene technology to the most robust and easy to transfect cells. In addition, reporter gene technology monitors gene expression as opposed to protein production. The vision of this proposal is to develop novel and transformative bio-microsystems for non-invasive, dynamic monitoring of protein production in difficult-to-transfect primary cells. This proposal will focus on the analysis of leukocytes and will develop biosensors and microsystems for monitoring cytokine production of individual T-lymphocytes. The interdisciplinary team of investigators will provide surface engineering and microfabrication-based solutions to challenges that currently confound single cell analysis, including: 1) creating high-density single cell arrays, 2) integrating multi-analyte sensors with single cells, 3) co-localizing sensing elements with single cells to ensure high local concentration of secreted metabolite. The novel biosensors to be developed in this proposal will be translatable to other cellular systems (e.g. circulating as well as anchorage-dependent cells) and will be broadly applicable in bioengineering, biotechnology and life sciences fields.The interdisciplinary collaboration of researchers with a diverse expertise in this project provides a unique opportunity and framework for interdisciplinary education and training of secondary school through postdoctoral students at the frontiers of engineering and the life sciences.