Multiplexed analysis of genes and of metal ions using enzyme/DNAzyme amplification machineries

The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg(2+)-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag(+), Hg(2+)). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic acid scaffolds that include recognition sites for the two genes and replication tracks that yield the nicking domains for Nt.BbvCI and two different Mg(2+)-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg(2+)-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F1 and/or F2 as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TP53 genes correspond to 0.1 nM. Similarly, two different nucleic acid scaffolds that include Ag(+)-ions or Hg(2+)-ions recognition sequences and the replication tracks that yield the Nt.BbvCI nicking domains and the respective Mg(2+)-dependent DNAzyme sequences are implemented as nicking/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds.