In this work, a label-free DNAzyme biosensor was proposed for the detection of oligonucleotides related to a hepatitis B virus (HBV) DNA segment. The novel DNAzyme biosensor was based on the electrochemical reduction of graphene oxide–carboxyl multi-walled carbon nanotube composites (GO–CMWCNTs) and the electrodeposition of gold nanoparticles (AuNPs) on a glassy carbon electrode (GCE). The thiolate hairpin capture probes with a caged G-quadruplex configuration were self-assembled on AuNPs through the well-known Au–thiol binding. Only after hybridization with target DNA, the hairpin configuration could be opened and released the particular guanine-rich nucleic acid sequences which could assemble to G-quadruplexes. In the presence of hemin and K+, the hemin/G-quadruplex DNAzyme was generated on the electrode surface, triggering the electrochemical H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). With the advantages of amplification effects of AuNPs and hemin/G-quadruplex, a linear concentration range from 10 pM to 10 nM with a limit of detection of 0.5 pM was obtained for the target DNA. This method exhibited excellent discrimination of target DNA from one or two-base mismatched DNA and non-complementary DNA sequences. In addition, the proposed biosensor was successfully applied to the determination of target DNA in human serum samples.
