DNAzyme-SYBR Green I Chemiluminescent System For High Signal-to-Background Ratio Sensing

G-quadruplex DNAzyme,formed by hemin and G-rich sequence,is widely used in biosensing due to its similar catalytic action with horseradish peroxidase?HRP?.For instance,G-quadruplex DNAzyme can catalyze H₂O₂ to oxidize luminol or2,2'-diazobis3-ethylbenzothiazoline-6-sulfonicacid?ABTS?andobtain chemiluminescent or visual signals?ABTS.⁺?.In some publications,hemin-aptamer?a G-rich sequence?was deliberately splited into two fragments which can reassemble into a G-quadruplex DNAzyme by the recognition of probe DNA with target DNA.Furthermore,quantitative detection of different DNAs can also be achieved by changing the recognition regions of probe DNA.These above methods have advantages of label-free,simple operation.Also,there are disadvantages such as high background and low signal-to-background ratio,which have positive influence on detection of trace DNA.This work constructs a DNAzyme-SYBR Green I?SG?chemiluminescent sensor for label-free,sensitive detection of DNA with low background and high signal-to-background ratio.The main contents are as follows:1.A chemiluminescent sensor with high signal-to-background ratio based on DNAzyme-SG was established.According to experimental datas,we found SG can heavily increase the signal-to-background ratio?from 4.20 to 22.6?of DNAzyme sensing system.For validating the mechanism,chemiluminescence,fluorescence spectrometry and UV spectrophotometry are used to investigate the chemiluminescent kinetics,influence of different dyes to the catalytic activity of hemin and DNAzyme,function of SG in different chemiluminescent systems and so on were investigated by chemiluminescence,fluorescence and absorption spectrum.The method that SG inhibits the catalytic activity of hemin was demonstrated by Michaelis constant?K_m?and maximum reaction rate(?_max)of catalytic reaction and the fluorescent spectrum of SG interacting with Hemin and the effects of compounds containing-SH and-NR groups.We also investigated the variation of chemiluminescent intensity when SG embeds in dsDNA and G-quadruplex DNAzyme forms and illustrated the SG-DNAzyme signal recovery mechanism.Based on the experimental data,we can conclude that the sulfydryl?–SH?of SG can efficiently decrease the catalytic activity of hemin by coordinating with Fe³⁺in the center of hemin.However,the catalytic signal will obviously increase in the prence of target DNA due to the combination of G-quadruplex and hemin,lower free SG concentration,and the intermolecular photocatalysis,etc..2.The DNAzyme-SG chemiluminescent sensor with high signal-to-background ratio was applied to detection of p53 gene.Factors such as G3 and G9 probe concentration,hemin concentration,SG concentration,DNA incubation temperature,the time of DNA incubation,and the time of SG incubation in the system were optimized.Under the optimal condition,the concentration of p53 gene in the range from 5.0 pM to 5.0 nM were linearly dependent on chemiluminescent signals to background ratio,with a detection limit of approximately 2.0 pM based on 3?.This sensor has good precision with RSD of 3.0%and excellent selectivity in DNA sensing.Compared with other nucleic acid determination methods,the present method has the advantages of free label,no amplification procedure,simple operation.The detection limit of detection?LOD?is compared to some signal amplification methods.The present method has the potential to apply to the detection for real samples.