Enzymatic cascade based fluorescent DNAzyme machines for the ultrasensitive detection of Cu(II) ions.
A novel enzymatic cascade based fluorescent DNAzyme machine has been developed for the amplified detection of copper (Cu(2+)) 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 Cu(2+) 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 Cu(2+) ions act as the activator for enzymatic cascade amplification generating multiple duplex structures in the nascent product. An increasing fluorescence is observed with increasing Cu(2+) ions concentration. A good nonlinear correlation (R=0.9997) was obtained between fluorescence intensity and the cubic logarithm of the Cu(2+) 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.
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China. Electronic address: jinglin_he@hotmail.com.
Jing-Lin
JL
He
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China.
Shuang-Li
SL
Zhu
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China.
Ping
P
Wu
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China.
Pan-Pan
PP
Li
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China.
Ting
T
Li
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, PR China. Electronic address: zhongcao2004@163.com.
Zhong
Z
Cao
D015374:Biosensing Techniques / Q000379:methods*; D003300:Copper / Q000032:analysis* / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D021881:DNA, Catalytic / Q000737:chemistry*; D007477:Ions; D009097:Multienzyme Complexes / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D021881:DNA, Catalytic; D007477:Ions; D009097:Multienzyme Complexes; D003300:Copper; D004247:DNA
Copper ions; DNAzyme machine; Endonuclease; Exponential amplification; Polymerase