Nanoshell sensors for cellular analysis

 描述（由申请人提供）：糖尿病的发病率在美国已达到流行病的比例，并且在可预见的未来将在所有年龄组和种族中继续快速增加。由此产生的社会成本，如医疗保健、工资损失等，是惊人的，生活质量的下降是不可估量的。2型糖尿病主要表现为两类，胰岛素分泌不足和胰岛素抵抗增强。在非糖尿病患者中，升高的血清葡萄糖刺激胰腺β细胞的胰岛素分泌并降低胰腺β细胞的胰高血糖素分泌。葡萄糖刺激的胰岛素分泌是一个复杂的过程，通过细胞内复杂的信号通路进行调节。细胞信号传导的动力学和相应的胰岛素释放对于血清葡萄糖的正常调节至关重要，然而，由于缺乏传感技术，许多细胞信号的检测是不可能的。最近的研究已经“重新发现”了β细胞在调节血清葡萄糖中的重要性，然而关于β细胞中代谢信号传导的动力学了解得少得多。更好地了解这两种细胞类型内的细胞内信号传导和相应的调节释放对于阐明分泌异常的根本原因以及糖尿病发病和进展中的相应作用至关重要。该提案的主要重点是开发适当的能力，以监测β-细胞和β-细胞中的代谢和碳水化合物衍生信号，并促进识别可能导致糖尿病的关键分子差异。我们将开发，表征和利用一种高度稳定的，多孔的磷脂结构，具有增强的质量传输能力，用于检测缺乏内在光学或电化学活性的细胞内调节剂。磷脂支架是用钙来制备的。5 nm厚的聚合磷脂膜，其中引入了尺寸选择性孔。多孔膜类似于透析膜，并且对小分子具有高渗透性，而与电荷无关，但保留/排除大分子量物质。这种架构将允许新的酶和荧光报告化学用于细胞内测量迄今无法检测的分析物。我们将通过研究新的聚合物稳定方案来优化多孔膜的形成，设计将生物可利用的传感器递送到细胞中的策略，并利用为丙酮酸盐设计的传感器， ATP，K+，葡萄糖和肌醇六磷酸，关键的β-细胞信号，其作用在β-细胞中定义较少，以研究这两种重要细胞类型中的代谢信号动力学。一旦完全开发，多孔脂质结构可以证明对于许多其他应用（包括大分子药物递送等）是有价值的。

 DESCRIPTION (provided by applicant): The incidence of diabetes mellitus has reached epidemic proportions in the U.S. and will continue to increase rapidly across all age group and ethnicities for the foreseeable future. The resulting cost to society via health care, lost wages, etc. is staggering and the decreased quality of life is immeasurable. Type 2 diabetes is primarily manifest in two categories, insufficient insulin secretion and enhanced insulin resistance. In non-diabetics, increased serum glucose stimulates insulin secretion from the pancreatic -cell and decreases glucagon secretion from the pancreatic -cell. Glucose-stimulated insulin secretion is a complex process that is regulated via complex signaling pathways within the cell. The dynamics of cellular signaling, and corresponding insulin release, are critical to normal regulation of serum glucose, however, detection of many cellular signals is not possible due to a dearth of sensing technologies. Recent studies have "rediscovered" the importance of the -cell in regulating serum glucose, however much less is understood regarding the dynamics of metabolic signaling in -cells. A better understanding of intracellular signaling and corresponding regulated release within both of these cell types is of paramount importance to elucidate the root causes of secretory abnormalities and the corresponding roles in the onset and progression of diabetes. The primary focus of this proposal is to develop suitable capabilities to monitor metabolic and carbohydrate-derived signals in - and -cells, and to facilitate identification of key molecular differences that may contribute to diabetes. We will develop, characterize and utilize a highly-stable, porous phospholipid architecture with enhanced mass transport capabilities for detection of intracellular regulators that lack intrinsic optical or electrochemical activity. Phospholipid scaffolds are prepared with ca. 5 nm thick polymerized phospholipid membranes into which size selective pores are introduced. The porous membranes are analogous to dialysis membranes and are highly permeable to small molecules irrespective of charge but retain/exclude large molecular weight species. This architecture will allow novel enzymatic and fluorescent reporter chemistries to be used for intracellular measurements of heretofore undetectable analytes. We will optimize the formation of porous membranes via investigation of novel polymer stabilization schemes, devise strategies to deliver bioavailable sensors into the cell and utilize sensors designed for pyruvate, ATP, K+, glucose and inositol hexakisphosphate, key -cell signals whose roles are less defined in the -cell, to investigate metabolic signaling dynamics in these two important cell types. Onc fully-developed, porous lipid architectures may prove valuable for a host of other applications including large molecule drug delivery, etc.