Charge-coupled device operated in a time-delayed integration mode as an approach to high-throughput flow-based single molecule analysis.
Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.
Emory
Jason M
JM
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Soper
Steven A
SA
D004247:DNA / Q000032:analysis*; D044085:Microfluidics / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D004247:DNA