Real time electrochemical monitoring of PCR amplicons using electroactive hydrolysis probe

A novel electrochemical DNA detection principle based on a hydrolysis DNA probe labeled with an electro-active indicator was developed. This strategy was further utilized to realize an electrochemical real-time PCR (ERT-PCR) technique which requires no probe immobilization step. Inspired by the Taqman® probe based fluorescence assay, our approach utilizes sequence-specific hydrolysis oligonucleotide probe modified with electroactive tags (also called eTaq probe). Upon the nucleotide extension step during the PCR, E-tagged nucleotides closer to the 5′ end of the hydrolysis probe would be cleaved away one-by-one as a result of the 5′ to 3′ exonuclease activity of the DNA polymerase. Unlike the negatively-charged hydrolysis probe, the released e-tagged mono- or short-nucleotide, because of the less extent of electrostatic repulsion with a negatively charged substrate surface, would diffuse freely to the negatively charged electrode (e.g. indium tin oxide) contributing to an increase of electrochemical signal. Experimental investigations demonstrate the cycle-by-cycle electrochemical signal enhancement. This new ERT-PCR method provides higher specificity and multiplexing capability and is a significant advancement towards the application of ERT-PCR in portable DNA-analysis.

► Unlike the fluorescence based real time PCR, this is a new electrochemical real-time PCR method. ► This method utilizes a sequence-specific probe modified with electroactive reporters. ► This method requires no probe immobilization step and it offers a platform highly compatible for multiplexed detection. ► This approach could soon rival with the fluorescence-based real-time PCR method in terms of sensitivity and specificity.