Microchip DNA electrophoresis with automated whole-gel scanning detection.
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abstract
Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment.
