IDBR: Ultrahigh Dynamic Range Fluorescence Detection for Capillary Electrophoresis

University Of Notre Dame, Notre Dame IN

Investigators

Abstract

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.