Triggered Polycatenated DNA Scaffolds for DNA Sensors and Aptasensors by a Combination of Rolling Circle Amplification and DNAzyme Amplification

摘要

The concept of triggered polycatenated DNA scaffolds hasnbeen elegantly introduced into ultrasensitive biosensingnapplications by a combination of rolling circle amplificationn(RCA) and DNAzyme amplification. As compared to traditionalnmethods in which one target could only initiate thenformation of one circular template for RCA reaction, in thenpresent study two species of linear single-stranded DNAn(ssDNA) monomers are self-assembled into mechanicallyninterlocked polycatenated nanostructures on capture probetaggednmagnetic nanoparticles (MNPs) only upon the introductionnof one base mutant DNA sequence as initiator fornsingle-nucleotide polymorphisms (SNPs) analysis. The resultantntopologically polycatenated DNA ladder is furthernavailable for RCA process by using the serially ligatedncircular DNA as template for the synthesis of hemin/GquadruplexnHRP-mimicking DNAzyme chains, which act asnbiocatalytic labels for the luminol-H2O2 chemiluminescencen(CL) system. Notably, the problem of high backgroundninduced by excess hemin itself is circumventednby immobilizing the biotinylated RCA products on streptavidin-nmodified MNPs via biotin-streptavidin interaction.nSimilarly, a universal strategy is contrived by substitutedlynemploying aptamer as initiator for the constructionnof polycatenated DNA scaffolds to accomplish ultrasensitivendetection of proteins based on structure-switchingnof aptamer upon target binding, which is demonstratednby using thrombin as a model analyte in this study.nOverall, with two successive amplification steps and onenmagnetic separation procedure, this flexible biosensingnsystem exhibits not only high sensitivity and specificitynwith the detection limits of SNPs and thrombin as lownas 71 aM and 6.6 pM, respectively, but also excellentnperformance in real human serum assay with no PCRnpreamplification for SNPs assay. Given the unique andnattractive characteristics, this study illustrates the potentialnof DNA nanotechnology in bioanalytical applicationsnfor both fundamental and practical research.