DETECTION OF ANALYTES SECRETED FROM SINGLE CELLS

拟议研究的目标是开发新的方法，用于 单细胞激素分泌的检测和测量。 这种方法需要两种能力来恢复分泌物质在 单细胞水平和测量目标分析物的能力 极低的水平。BioTraces专有的多光子探测 (MPD)技术允许检测微小(小于10-18摩尔) 生物材料的数量。这种单元化的能力将是 与乔治医院的微透析取样器相结合 华盛顿大学医学中心(GWUMC)将量化 单细胞水平的激素分泌。 GWUMC微透析仪将改装为 适应能够在单细胞水平上工作的透析探头。 从这个单一仪器获得的样品将用MPD进行分析 增强型高效液相色谱(HPLC)和 BioTraces专有的SuperIRMA免疫分析协议。在第一阶段 该系统将用于对大鼠胰岛素的合成进行量化 人成纤维细胞中的胰腺细胞和胰岛素样生长因子-I。 对这些目标的MPD增强高效液相和SuperIRMA检测将是 已开发，其准确性和精确度将在阶段进行验证 我。 潜在的商业应用 从单个活细胞中获取和分析样本的能力是 在发病机制和治疗研究中的巨大重要性 许多疾病，包括糖尿病。这里描述的系统将 为研究细胞内的多肽生产提供了通用工具 水平，因此将成为了解 细胞水平上的生理功能和疾病反应。

The goal of the proposed research is to develop novel methods for the detection and measurement of hormone secretion from a single cell. Such methods require the both ability to recover secreted materials at the single cell level and the capability to measure the target analytes at extremely low levels. BioTraces' proprietary Multi Photon Detection (MPD) technique permits detection of minute (less than 10-18 mole) quantities of biological material. This unitque capability will be combined with micro-dialysis sampling apparatus at George Washington Unviersity Medical Center (GWUMC) to quantify hormone secretion at the single cell level. The GWUMC micro-dialysis apparatus will be modified to accommodate dialysis probes capable of work at the single cell level. Samples obtained from this single apparatus will be anlyzed by MPD enhanced high performance liquid chromatography (HPLC) and by BioTraces' proprietary SuperIRMA immunoassay protocol. In Phase I this system will be used to quantify the synthesis of insulin from rat pancreatic cells and insulin-like growth factor-I from human fibroblasts. MPD-enhanced HPLC and SuperIRMA assays for these targets will be developed and their accuracy and precision will be validated in Phase I. POTENTIAL COMMERCIAL APPLICATIONS The ability to obtain and analyze samples from single living cells is of immense importance in the study of the pathogenesis and treatment of many diseases, including diabetes. The system porposed herein will provide a generic tool for studying peptide production at the cellular level, and thus will be an indispensable tool for understanding physiological function and disease response at the cellular level.