High-sensitivity microvolume UV absorption spectrometry for routine analysis of small-volume biological samples

Abstract

Substantial improvement of microvolume UV absorption spectrometry in sensitivity, robustness and ease of operation was achieved for routine biological applications. A unique microtubing-based absorption cell (208 μm internal diameter) featuring enhanced light transmission with a liquid core waveguide technique provided dramatically enhanced absorption sensitivity, proportional to the extended path length (50 mm, from the typical 1 mm), while robust measurement performance was attained by implementation of preventive measures against bubble trapping along the light path. For pBR322 plasmid DNA, absorbance at 260 nm was reliably measurable down to 0.1 ng/μl with repeatability typically 2–3% RSD. The detection limit was 0.03 ng/μl dsDNA, far lower than the current state-of-the-art ∼1 ng/μl. Sample consumption for each measurement was 2.4 μl. Automated operation implemented for the first time in microvolume spectrophotometry facilitated the ease in handling with small-volume biological samples.