Enzymatic cascade based fluorescent DNAzyme machines for the ultrasensitive detection of Cu(II) ions

• This is the first attempt to combine the self-cleaving DNAzyme with multi-enzyme systems.
• The enzymatic cascade based DNAzyme machines lead to exponential amplification.
• The Cu2+ ions act as the activator for enzymatic cascade reaction.
• The nonlinear response is attributed to extra amplification of enzymatic cascade.

A novel enzymatic cascade based fluorescent DNAzyme machine has been developed for the amplified detection of copper (Cu2+) ions. This is the first attempt to carry out the combination of the self-cleaving DNAzyme and the polymerase/endonuclease reaction cycles involving cleaved substrate extension. In the presence of Cu2+ ions, the enzyme strand carries out catalytic reactions to hydrolytic cleavage of the substrate strand. The cleaved DNAzyme substrates act as primers and trigger the Klenow Fragment polymerization. Nb.BbvCI endonuclease cuts the double-stranded niking site and thus opens a new site for a new replication. The replication regenerates the complete dsDNA to initiate another cycle of nicking, polymerization and displacement. Finally the fluorescence dye, SG, inserts into the DNA double helix to generate a distinguishable fluorescence enhancement. The Cu2+ ions act as the activator for enzymatic cascade amplification generating multiple duplex structures in the nascent product. An increasing fluorescence is observed with increasing Cu2+ ions concentration. A good nonlinear correlation (R=0.9997) was obtained between fluorescence intensity and the cubic logarithm of the Cu2+ ions concentration over the range 0.50–200 nM. This nonlinear response phenomenon results in an efficient improvement of the sensitivity of our current proposed assay. The activation of such enzymatic cascades through analyte–DNAzyme interactions is not only valuable to activate the cooperation of enzyme networks, but also has a substantial impact on the development of amplified DNAzyme sensors.