IDBR: Ultrahigh Dynamic Range Fluorescence Detection for Capillary Electrophoresis

IDBR：用于毛细管电泳的超高动态范围荧光检测

• 批准号: 0963826
• 负责人: Norman Dovichi
• 金额: $ 13万
• 依托单位: University of Notre Dame
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0963826&HistoricalAwards=false
• 关键词: IDBR; Ultrahigh; Dynamic; Range; Fluorescence

Laser-induced fluorescence is a technique used for the study of minute amounts of fluorescent material. The combination of capillary electrophoresis with ultrasensitive laser-induced fluorescence has proven to be a powerful tool in bioanalysis. As the preeminent example, the human genome was sequenced using capillary electrophoresis arrays.Dynamic range refers to the analyte concentrations that can be measured reliably. It is limited for low signals (low concentrations) by background noise, and at high signal (high concentration) by detector saturation or other sources of non-linearity. A number of bioassays would benefit from wider dynamic range. Examples of such systems include the study of rare post-translational modifications of proteins, characterization of the ultralow methylation status of DNA, detection of protein serum markers at very low concentrations, detection of rare circulating cancer cells in the presence of a very large excess of normal cells, enzyme kinetics at extreme substrate excess, study of specific metabolic cascades, characterization of binding events across a very wide concentration range, and detailed description of errors in transcription/translation.In this project a laser-induced fluorescence detector will be developed for capillary electrophoresis, with a 12 order of magnitude dynamic range. This detector provides over 100,000,000 times wider dynamic range than currently available commercial instruments. The instrument is based on two modules. Roughly 99% of the fluorescence signal will be sent to a single-molecule detection module, which will be used to quantitate from 1-1,000 analyte molecules. The remaining fluorescence signal will be sent to high-dynamic range module that will be used to quantitate from 1000 to 1,000,000,000,000 analyte molecules. The broader impacts of the project include enabling the detection of minute metabolic products generated from a huge excess of fluorescently-tagged substrate, thereby enabling advances in biological research. Results of the work will be made available to the community through publications and open-source web-based resources.

激光诱导荧光是一种用于研究微量荧光材料的技术。毛细管电泳与超灵敏激光诱导荧光的结合已被证明是生物分析中的有力工具。作为一个突出的例子，人类基因组测序使用毛细管电泳阵列。动态范围是指分析物的浓度，可以可靠地测量。对于低信号（低浓度），它受到背景噪声的限制，对于高信号（高浓度），它受到检测器饱和或其他非线性源的限制。许多生物测定将受益于更宽的动态范围。这样的系统的实例包括蛋白质的罕见翻译后修饰的研究，DNA的超低甲基化状态的表征，在非常低浓度下蛋白质血清标志物的检测，在非常大量过量的正常细胞存在下罕见循环癌细胞的检测，在极端底物过量下的酶动力学，特定代谢级联的研究，在这个项目中，将开发一种用于毛细管电泳的激光诱导荧光检测器，其动态范围为12个数量级。该探测器提供的动态范围比目前可用的商业仪器宽100，000，000倍。该仪器基于两个模块。大约99%的荧光信号将被发送到单分子检测模块，该模块将用于定量1- 1，000个分析物分子。剩余的荧光信号将被发送到高动态范围模块，该模块将用于定量1000至1，000，000，000，000个分析物分子。该项目更广泛的影响包括能够检测由大量过量的荧光标记底物产生的微小代谢产物，从而促进生物研究的进展。工作成果将通过出版物和开放源网络资源向社区公布。