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.
" funded the development of a state-of-the-art laser-induced fluorescence detector for capillary electrophoresis. The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam-splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. The instrument demonstrates nine orders of magnitude dynamic range, and has been used to characterize glycophingolipid metabolism in single neurons. In addition, a new design for a sheath-flow cuvette was developed; this design is inexpensive and simple, which should facilitate the wider use of this technology in capillary electrophoresis and other fields. Two publications have resulted from this grant: 1. Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters. Dada OO, Essaka DC, Hindsgaul O, Palcic MM, Prendergast J, Schnaar RL, Dovichi NJ.Anal Chem. 2011 Apr 1;83(7):2748-53. 2. Simplified sheath flow cuvette design for ultrasensitive laser induced fluorescence detection in capillary electrophoresis.Dada OO, Huge BJ, Dovichi NJ. Analyst. 2012; 137: 3099-101.
