Colorimetric Detection of Copper(II) Ion Using Click Chemistry and Hemin/G-Quadruplex Horseradish Peroxidase-Mimicking DNAzyme

G-quadruplex-forming sequence can be formed through a copper(I) ion (Cu+)-catalyzed click chemistry between azide- and alkyne-modified short G-rich sequences in aqueous solution, eliminating immobilization and washing steps of conventional assays. The source for Cu+ was generated from the reduction of Cu2+ with the reductant of sodium ascorbate. In the presence of hemin and K+, the self-assembly of hemin/G-quadruplex structure has the activity of horseradish peroxidase (HRP), which can catalyze its colorless substrate tetrazmethyl benzidine (TMB) into a colored product. Hence, the concentration of Cu2+ can be evaluated visually for qualitative analysis according to the color change of the solution, and the optical density (OD) value of the resulting solution at 450 nm was also recorded using a microplate reader for quantitative analysis.