Hybridization-based sensor with large dynamic range for detection of circulating tumor DNA in clinical samples

In a liquid biopsy approach, targeted mutation analysis of circulating tumor DNA (ctDNA) is a valuable tool for diagnosis, monitoring and personalization of therapy. The ctDNA usually makes up only a small fraction of the total circulating free DNA (cfDNA), and ctDNA often only differs from cfDNA at a single nucleotide. This sets strong requirements on the analytical performance of hybridization-based biosensors, which is the focus of this paper. We use clinical samples and apply the concept of wild-type target depletion. Along with this, we develop an accurate thermodynamic theory for the competitive hybridization and use it for selecting optimal experimental conditions and for data analysis. The result is a biosensor with improved quantification of ctDNA mutations, both the sensitivity and dynamic range are improved by an order of magnitude. As reference techniques, we used a clinically-validated real-time PCR assay and digital PCR for absolute quantification. Our approach can be applied to a broad range of hybridization-based biosensors, providing a robust and effective method to improve the performance of existing biosensors.