Decoding circulating nucleic acids in human serum using microfluidic single molecule spectroscopy.

Circulating nucleic acid (CNA) has been the focus of recent research as a noninvasive source of biomarker candidates. Among these markers, DNA fragment size has shown promise for discerning the source of CNA molecules in cancer and prenatal diagnostics. We have developed a one-step assay for analyzing circulating DNA size and quantity directly in human serum. Microfluidic cylindrical illumination confocal spectroscopy and fluorescence burst size analysis are used to individually count and size fluorescently-labeled CNA molecules as they are driven through a microfluidic constriction. First, single molecule sizing was performed on lambda Hind III digest DNA to obtain a size calibration curve. A linear relation between DNA length and burst size was seen from 564 bp to 27.5 kbp. Subsequently, the single molecule assay parameters were optimized. Finally, DNA sizing analysis was 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, and without the need for DNA isolation or enzymatic amplification steps. This demonstrates that microfluidic single molecule spectroscopy can be a rapid, facile, and inexpensive alternative to the established PCR-based methods that have been used near exclusively for CNA analysis.