A homogeneous digital biosensor for circulating tumor DNA by the enumeration of a dual-color quantum dot complex

Monitoring ctDNA in blood is important for cancer management. Here, a one-step single particle counting approach was developed for directly quantifying ctDNA in plasma. Hairpin DNA containing a triple helix stem was immobilized onto QD 585 as a probe. The hairpin was opened by the target, and therefore hybridized with assistant DNA on QD 655, resulting in an aggregate of QD 585 and QD 655. The two-color QD aggregate was regarded as the target. Observed under a single particle transmission grating-based spectral microscope, the two-color QD aggregate was distinguished by a unique spectral pattern of two first-order streaks, and it was counted. The difference in the responses of the probes to perfect-match DNA, single-base mismatch DNA, and non-match DNA indicated that the probe had sufficient single-base discrimination capabilities. The success in plasma recovery tests demonstrated the feasibility of carrying out the direct detection of ctDNA in plasma.