Click DNA cycling in combination with gold nanoparticles loaded with quadruplex DNA motifs enable sensitive electrochemical quantitation of the tuberculosis-associated biomarker CFP-10 in sputum

Abstract

An electrochemical aptamer-based assay is described for the determination of CFP-10 which is an early secretary biomarker of Mycobacterium tuberculosis. CFP-10 is specifically captured by its aptamer and then induces a DNA cross-linking click reaction, the release of CFP-10, and an amplification cycle of repeated CFP-10 release. This mechanism (with dual amplification via DNA click and target release cycle) causes more and more CFP-10 Apt strands on the electrode surface to expose their 5' overhang and to hybridize with the DNA complexes linked to the gold nanoparticles (AuNPs). Consequently, large amounts of AuNPs, each loaded with a number of quadruplex DNA motifs, can be bound on the electrode surface and remarkably enhance the signal. Under optimal conditions, the method has a detection limit as low as 10 pg.mL^-1 of CFP-10. The method was successfully applied to the diagnosis of M. tuberculosis in sputum. Graphical abstract Schematic representation of an electrochemical CFP-10 (10-kDa culture filtrate protein) assay using click DNA cycling in combination with gold nanoparticles loaded with quadruplex DNA motifs. Click chemistry reaction between Dibenzocyclooctyne (DBCO)-DNA and azido-DNA can liberate the CFP-10 antigen for the next cycle, which can be viewed as the first amplification step. G-quadruplex-based DNAzyme is formed due to the guanine-rich sequences of DNA S1, which can be viewed as the second amplification step.

Keywords: Aptasensor; AuNPs; Azido group; DNA click ligation; Dual amplification strategy; Electrochemical method; G-quadruplex-hemin complex; H2O2; Hydroquinone; Mycobacterium tuberculosis.