ctDNA Detection Based on DNA Clutch Probes and Strand Exchange Mechanism

Abstract

Circulating tumor DNA (ctDNA), originating directly from the tumor or circulating tumor cells, may reflect the entire tumor genom and has gained considerable attention for its potential clinical diagnosis and prognosis throughout the treatment regimen. However, the reliable and robust ctDNA detection remains a key challenge. Here, this work designs a pair of DNA clutch separation probes and an ideal discrimination probes based on toehold-mediated strand displacement reaction (TSDR) to specifically recognize ctDNA. First, the ctDNAs were denatured to form ssDNAs, the pair of DNA clutch separation probes [one of which modified onto the magnetic nanoparticles (MNPs)] are used to recognize and hybridize with the complemental chains and prevent reassociation of denatured ssDNAs. The complemental chains are removed in magnetic field and left the wild and mutant ssDNA chains in the supernatant. Then, the TSDR specificity recognizes the target mutant sequence to ensure only the mutated strands to be detection. The proposed assay exhibited good sensitivity and selectivity without any signal amplification. The proposed assay displayed a linear range from 2 to100 nM with a limit of detection (LOD) of 0.85 nM, and it was useful for ctDNA biomedical analysis and clinic theranostic.

Electrochemical Biosensors for Circulating Tumor DNA Detection

“…For the completeness of the discussion on ctDNA biosensors, the optical sensing methods of ctDNA are briefly presented as follows. Chang et al [ 58 ] proposed a fluorescence polarization method to detect ctDNA based on DNA dissociation probe and chain exchange mechanism and used toehold-mediated strand displacement reaction (TSDR) to identify the specific binding of probes to target mutated DNA to generate fluorescence for qualitative analysis. Jin et al [ 59 ] designed and synthesized a water-soluble cationic fluorescent probe and found that fluorescence intensity was linearly proportional to ctDNA concentration at a certain concentration.…”

Electroanalytical Biosensors for Circulating Tumor DNA Detection—A Brief Review

“…Chang et all. [51] proposed a fluorescence polarization method to detect ctDNA based on DNA dissociation probe and chain exchange mechanism, and used toehold-mediated strand displacement reaction (TSDR) to identify the specific binding of probes to target mutated DNA to generate fluorescence for qualitative analysis. Jin J et all.…”

“…Level Reference Electrochemical method based on DNA probe fM [34], [38], [39], [41], [42] Electrochemical method based on antigen antibody fM [51], [52] Fluorescence method pM [57],[58] Amplified colorimetric method pM [59]…”

The Growing Impact of Micro/Nanomaterial‐Based Systems in Precision Oncology: Translating “Multiomics” Technologies

“…In a recent approach, Chang et al developed a magnetic nanoparticle-based separation technique for BRAF ctDNA purification, and achieved a detection limit of 0.85 × 10 −9 m in serum samples. [39] In this method, magnetic nanoparticles functionalized with clutch separation probes were designed to bind to complementary strands and prevent reassociation of denatured ssDNA sequences. After removal of complementary strands by magnetic separation, toehold-mediated strand displacement reaction was further used to specifically recognize BRAF target mutant sequences for fluorescence detection.…”