Application of the Hybridization Chain Reaction on Electrodes for the Amplified and Parallel Electrochemical Analysis of DNA

The hybridization chain reaction (HCR) is implemented for the development of amplified electrochemical DNA sensing platforms. The target analyte hybridizes with a probe oligonucleotide-functionalized electrode and triggers on the HCR process in the presence of the hairpins H_A and H_B. The formation of the analyte-triggered HCR chains is followed by Faradaic impedance spectroscopy or chronocoulometry using Fe(CN)₆^3-/4- or Ru(NH₃)₆³⁺ as redox labels, respectively. By using two different probe-functionalized electrodes and a mixture of four hairpins, H_A:H_B and H_C:H_D, the parallel analysis of two analytes is demonstrated. Through the structural design of the hairpin structures to include caged G-quadruplex subunits, the analyte/probe hybrid associated with the electrode triggers on the HCR process, leading to G-quadruplex-functionalized HCR chains. The association of hemin to the matrix yields electrocatalytic hemin/G-quadruplex units that provide a secondary amplification path for the detection of DNA through an electrocatalyzed reduction of H₂O₂. The system allows the detection of the analyte DNA with a detection limit corresponding to 0.2 nM.