| A 32-capillary direct-reading multi-wavelength spectrometer was developed for separation and detection of fluorescently labeled biological macromolecules using capillary electrophoresis with laser-induced fluorescence detection. This spectrometer provides high sample throughput by analyzing 32 different samples simultaneously. In addition, the spectrometer is able to measure the spectral profile of a fluorescent dye. Discrimination of macromolecules with different labels is achieved by the comparison of the emission profiles of the labels. The spectrometer was characterized by the limit of detection, and further examined by four-color DNA sequencing with capillary gel electrophoresis and peptide analysis with capillary zone electrophoresis.;The excitation source of the spectrometer was a multi-line argon ion laser (488 nm, 514.5 nm). The laser beam was introduced into a 32-capillary sheath-flow cuvette to simultaneously excite fluorescence from the sample streams of 32 capillaries. The fluorescence was then color-dispersed by the dispersion optics, and the spectra were detected by a CCD camera which was controlled by the data acquisition software through a Macintosh computer.;The optical design of the dispersion optics was assisted by calculation and computer- modeling. The spectral dispersion was achieved by the use of two identical camera lenses (55 min f/1.4) and an F2 equilateral prism. The fluorescence light collected from the cuvette was collimated by the first lens, color-dispersed by the prism, and then imaged onto the detector by the second lens.;Data acquisition was performed with a software written in C. The software controls the exposure and data transfer of the CCD camera, writes spectral data onto hard disk, and allows the user to select the effective exposure regions and to monitor one spectral channel of any selected capillary. Data analysis was also performed automatically by using the procedures written in Igor Pro.;In addition to the instrument development, separation conditions of DNA sequencing with linear polyacrylamide were optimized using a 5-capillary instrument. Sequencing accuracy and read-length are increased by operating capillaries at high temperatures; about 600 bases can be sequenced within 2 h at a 150 V/cm electric field and 60°C in 5%T polyacrylamide. |
