Instrumentation for the analysis of biological molecules

Darren F Lewis
2003
This thesis describes the development of instrumentation designed for the analysis of biological molecules. Each instrument performs laser-based chemical analysis of minute quantities of analyte. Two instruments described in this work were built for use in the analysis of DNA, and a third instrument provides spectral analysis on biological particles derived from ambient aerosol. The DNA sequencing instrument development focused on several aspects of detector design, including automation, data
more » ... quisition timing, avalanche photodiode optimization, and data collection software development. The finalized DNA sequencing instruments were capable of robust routine operation and possessed detection limits between 130 and approximately 10,000 molecules of analyte in single color data collection mode. Linear-mode avalanche photodiode (APD) optimization required analysis of the noise produced under weakly illuminated conditions. APD noise contributions were evaluated for a series of different illumination conditions. Although APDs possess high quantum efficiencies, their internal amplification gain noise limits the device signal-to-noise ratio, and optimum settings require small APD gain with large transimpedance gain. Avalanche photodiodes are better suited to the detection of signals that are bandwidth limited if gain must be provided through external amplification. A biological cell analyzer was developed that allows simultaneous determination of native fluorescence and laser scatter for particle sizing. The detector could analyze individual analyte cells, and was useful for obtaining crude size information with photon shot noise dominated spectra on these cells. When populations of different cells were analyzed, population disparities allowed sub-populations to be discerned. This detection apparatus is at an early stage of development, but the method holds promise for future use.
doi:10.7939/r3-frk7-6p73 fatcat:cfwb3acvvndxxndopoi6cnqupa