Bulge-loop tuned entropy-driven catalytic reaction and tag-encoded barcodes for multiplexed mutation detection

Sensitive and specific detection of single nucleotide variants (SNVs) has great potential for disease diagnosis and prognosis. Hybridization probes based on DNA strand displacement have shown high SNV discrimination and programmability. However, it remains a challenge to balance the specificity and sensitivity of hybridization reactions due to the limited adjustability of probe sequences. Herein, we inserted bulge-loops in probes to fine-tune the thermodynamic changes of the entropy-driven catalytic reaction (EDC), significantly improving the selectivity (the DF of CAC>CGC mutation was promoted from 17.66832±2.25913–72.54±8.21) and responsiveness of SNV identification. In addition, oligonucleotides with chromophores (Tags) were used to impart microspheres with different fluorescence characteristics, to construct a set of convenient and flexible barcodes. Coupled with the bulge-loops tuned EDC probes and the Tag-encoded barcodes, the designed biosensor achieved higher specificity (DF=194.86±62.11) and lower signal leakage. Most importantly, it allowed simultaneous detection of low-abundance five-plexed EGFR mutations in the same system. This work provided insights into a rational design of DNA cascade reaction networks and advanced the construction of customizable multiplexed detection systems in the suspension array technology.