PCR-free, microfluidic single molecule analysis of circulating nucleic acids in lung cancer patient serum.

Circulating nucleic acid (CNA) has been the focus of much recent research, studied both as a diagnostic marker and as a marker for enrichment of diseased DNA. Among these markers, circulating DNA fragment size has shown promise for discerning the source of CNA molecules in cancer and prenatal diagnostics due to differences in average size between cancer vs. healthy or fetal vs. maternal DNA. We describe a 1-step assay for analyzing circulating DNA size and quantity directly in human serum that replaces complicated nested qPCR analysis. Microfluidic cylindrical illumination confocal spectroscopy and fluorescence burst size analysis were used to individually count and size fluorescently-labeled CNA molecules as they were driven through a microfluidic constriction. First, single molecule sizing was performed on λ Hind III digest DNA to obtain a size calibration curve. A linear relation between DNA length and fluorescent burst size was seen from 564 bp-23.1 kbp. Then, the single molecule assay was used to analyze an in vitro model of DNA fragmentation. Finally, DNA sizing analysis was successfully performed on serum samples from both early and late stage lung cancer patients. This assay was performed directly in patient serum using only a single reagent, a simple DNA intercalating dye. Furthermore, it eliminated the need for DNA isolation or enzymatic amplification. This demonstrates that microfluidic single molecule spectroscopy can be a rapid, facile, and inexpensive alternative to the established PCR-based methods.