Counting DNA molecules on a microchannel surface for quantitative analysis

Abstract

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/μL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 μL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis. © 2022 Elsevier B.V.