TdT combined with Cas14a for the electrochemical biosensing of NPC-derived exosomes

Abstract

In this work, the electrochemical biosensor based on the subtle combination of terminal deoxynucleotidyl transferase (TdT), CRISPR/Cas14a, and magnetic nanoparticles (MNPs) was developed for the detection of nasopharyngeal carcinoma (NPC)-derived exosomes. Due to the synergistic effect of the following factors: the powerful elongation capacity of TdT for single-stranded DNA (ssDNA) with 3-hydroxy terminus, the outstanding trans-cleavage ability of CRISPR/Cas14a specifcally activated by the crRNA binding to target DNA, and the excellent separation ability of MNPs, the developed electrochemical biosensor exhibited high sensitivity for the detection of NPC-derived exosome, with a linear range from 6.0 × 10² ∼ 1.0 × 10⁵ particles/mL and a limit of detection as lown as 80 particles/mL. In addition, this electrochemical biosensor successfully distinguished exosomes from NPC patients and healthy individuals. This electrochemical biosensor opens up a new pathway for the early diagnosis of NPC.

Abbreviations: NPC, Nasopharyngeal carcinoma; CRISPR/Cas, Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system; PAMs, Protospacer adjacent motifs; RCA, Rolling circle amplification; CHA, Catalytic hairpin assembly; LAMP, Loop-mediated isothermal amplification; TdT, Terminal deoxynucleotidyl transferase; SgRNA, Single guide RNA.