IDBR: Ultrahigh Dynamic Range Fluorescence Detection for Capillary Electrophoresis

激光诱导荧光是一种用于研究微量荧光材料的技术。毛细管电泳联用超灵敏的激光诱导荧光已被证明是生物分析中的一种强有力的工具。作为一个突出的例子，使用毛细管电泳阵对人类基因组进行了测序。动态范围指的是可以可靠地测量的分析物浓度。它在低信号(低浓度)时受到背景噪声的限制，在高信号(高浓度)时受探测器饱和或其他非线性源的限制。许多生物分析将受益于更大的动态范围。这些系统的例子包括研究罕见的蛋白质翻译后修饰，表征DNA的超低甲基化状态，在非常低的浓度下检测蛋白质血清标志物，在存在非常大量的正常细胞的情况下检测罕见的循环癌细胞，在极端底物过剩时的酶动力学，研究特定的代谢级联反应，在非常宽的浓度范围内表征结合事件，以及详细描述转录/翻译中的错误。在这个项目中，将开发一种激光诱导荧光检测器用于毛细管电泳，动态范围为12个数量级。该探测器提供的动态范围是目前商用仪器的100,000,000倍以上。该仪器由两个模块组成。大约99%的荧光信号将被发送到单分子检测模块，该模块将用于对1-1000个分析物分子进行定量。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到1,000,000,000,000个分析物分子。该项目的更广泛影响包括能够检测到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码的网络资源提供给社区。

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.