Microfabricated Biosensor Platform for Monitoring of Cell Metabolism

描述（申请人提供）：肝脏是人体重要的生物系统，在调节机体能量供应中起着关键作用。该器官维持葡萄糖释放和储存程序，以响应激素刺激，建立葡萄糖稳态。毒物对能量代谢的破坏具有严重的病理生理后果，并与糖尿病和肥胖有关。测量细胞代谢的传统方法需要大量的细胞数量，对代谢动力学的揭示很少，不允许细胞同时暴露于多种刺激下，也不允许研究多种细胞类型之间的相互作用。因此，传统的细胞培养/代谢物检测方法不适合监测稀缺细胞（如原代人肝细胞）和筛选候选药物和毒物对肝细胞的影响。本提案的目标是开发一种新型的实验室芯片生物传感器平台，用于体外监测肝细胞代谢。该平台的新颖性是基于我们将多种代谢物的微生物传感器与功能性微合成肝细胞培养物相结合的能力。细胞和传感元件的并置将通过制造聚乙二醇（PEG）水凝胶微结构来实现，该微结构具有细胞培养室和酶包封基质的双重功能。含酶的PEG水凝胶微室将建立在细胞培养底物的顶部，创建单独的可寻址的细胞培养单元，用于葡萄糖的电化学检测和过氧化氢（H2O2）的荧光检测。提出的生物传感器平台将提供几个优势，包括同时监测两种分析物，观察两种不同细胞表型的动态和互惠代谢相互作用，并利用少量细胞进行分析。微流体输送系统将与传感模块结合，以控制可溶环境的组成。一种集成功能肝细胞和微型生物传感器的新型微制造装置将成为研究肝毒物对肝功能影响的重要使能技术。肝脏是人体的代谢中心，在很大程度上负责解毒和能量调节功能。能量调节是指糖的消耗和产生的程序，如葡萄糖。能量调节失衡与糖尿病和肥胖密切相关。我们建议制造一种微芯片来监测肝细胞中的糖消耗。这种微芯片将用于确定有害物质如何影响肝脏及其调节体内糖水平的能力。

DESCRIPTION (provided by applicant): Liver is an important biological system that plays a key role in regulating energy supply of the body. This organ maintains a program of glucose release and storage in response to hormonal stimulation establishing glucose homeostasis. The disruption of energy metabolism by toxicants has serious pathophysiological consequences and is connected to diabetes and obesity. Traditional approaches for measuring cellular metabolism require large cell numbers, reveal little about metabolism dynamics, do not allow exposure of cells to multiple stimuli in parallel and do not permit to study the interplay between multiple cell-types cultured together. Thus, the traditional cell culture / metabolite detection approaches are not suited for monitoring scarce cells (e.g. primary human hepatocytes) and screening the effects of drug candidates and toxicants on the liver cells. The goal of this proposal is to develop a novel lab-on-chip biosensor platform for in vitro monitoring of liver cell metabolism. The novelty of the proposed platform is based on our ability to integrate micro-biosensors for multiple metabolites with functional microfabricated liver cell cultures. The juxtaposing of cells and sensing elements will be achieved by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures serving dual function as cell culture chambers and matrices for enzyme encapsulation. Enzyme-containing PEG hydrogel microchambers will be built on top of cell culture substrates, creating individually addressable cell culture units for electrochemical detection of glucose and fluorescence-based detection of hydrogen peroxide (H2O2). The proposed biosensor platform will offer several advantages including monitoring two analytes simultaneously, observing dynamic and reciprocal metabolic interactions of two distinct cell phenotypes, and utilizing small numbers of cells for analyses. A microfluidic delivery system will be incorporated with the sensing module to control composition of the soluble environment. A novel microfabricated device integrating functional liver cells with miniature biosensors will be an important enabling technology in the study of the effects of hepatotoxicants on liver function. PUBLIC HEALTH RELEVANCE Liver is the metabolic center of human body and is largely responsible for detoxification and energy regulation functions. Energy regulation refers to the program of consumption and production of sugars such as glucose. Imbalance in energy regulation is closely connected diabetes and obesity. We propose to build a microchip for monitoring sugar consumption in liver cells. This microchip will be used to determine how harmful substances affect the liver and its ability to regulate sugar levels in the body.