Electrochemiluminescence biosensor for HPV16 detection based on the adjusting of steric hindrance effect coupled with Exonuclease III amplification strategy

The effect of steric hindrance of electrode surface can affect the diffusion of the Ru(bpy)32+ toward electrode and thus in turn affect the ECL performance of the system. In this study, this character had been adopted to develop a biosensor for HPV DNA detection. Exonuclease III (Exo III) signal amplification strategy had been applied to realize signal amplification. First, hairpin probes (HP) was anchored on the surface of electrode as capture probes, HP can resistant to the hydrolyzation of Exo III due to its 3'-protruding termini. Without the target, a large amount of cDNA modified Au nanocages (AuNCs-cDNA) can hybridize with HP and connected to surface of electrode, weak ECL signals can be detected since Ru(bpy)32+ can not diffuse freely to the electrode surface because of the steric hindrance of AuNCs-cDNA. In the presence of the target, HP can hybridize with the target to form double-stranded DNA (dsDNA) with a blunt 3' terminus, due to the high preference of Exo III for cleaving dsDNA with a blunt 3' termini, HP in dsDNA was hydrolyzed, and the target which formed dsDNA was released to hybridize with another HP, inducing the Exo III assisted amplification strategy. Due to the reduction of HP on electrode surface, the amount of AuNCs-cDNA connected to the electrode surface become small, a high ECL signal can be detected. Under the optimal conditions, the ECL response of the system has a linear relationship with logarithm of target DNA concentration in the range of 10 fM to 100 pM, and a detection limit of 3.54 fM (S/N = 3). The proposed biosensor has high sensitivity and selectivity, which had been applied to the detection of target DNA in real sample and the satisfied results had been obtained. This system also can detect different targets by changing the DNA sequence easily.