Ratiometric Fluorescent Probes For Nucleic Acids Detection Based On DNA-Silver Nanoclusters

Ratiometric fluorescent probes achieve the output of ratio signals by introducing multiple fluorescent peaks,which can reduce the interference of environment and eliminate systematic errors,thereby improving the detection reliability.Although the developed ratiometric fluorescence methods have good detection performance,fluorescent probes need to be labeled and are limited by the anti-photobleaching properties of organic fluorescent dyes and the number of donor-acceptor pairs.With the development of nanotechnology,based on the development of novel fluorescent nanomaterials,ratiometric fluorescent probes not only can overcome such drawbacks,but also have the advantages of low cost,high biocompatibility,good water solubility,adjustable fluorescence spectrum,and easy functionalization.They have been widely used in biochemical analysis.Among them,"chameleon"DNA-silver nanoclusters?DNA-AgNC?have become new materials for ratiometric fluorescent probe design,because not only they have multiple fluorescent emission peaks,but also they are simple in synthesis,inexpensive,and flexible in design without complex fluorescent labeling.The occurrence and development of cervical cancer is associated with persistent infection of human papillomavirus?HPV?,which can be used for screening for cervical cancer by HPV DNA testing.In this paper,a ratiometric fluorescent probe was designed and synthesized using the"chameleon"DNA-AgNC to realize the detection of HPV DNA in human serum samples.The research content s include:1 Chameleon DNA-silver nanocluster ratiometric binary probes for detection of HPV-related DNAHerein,we described a ratiometric strategy based on"chameleon"DNA-silver nanocluster?DNA-AgNC?fluorescent binary probes.The strategy was then applied to detect high-risk human papillomavirus?HPV?DNA sequences,HPV-16.First,DNA-AgNCs were synthesized by a simple reduction method.The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs.Upon the addition of HPV-16 DNA,the yellow fluorescence of AgNCs was reduced greatly,whereas the red fluorescence of AgNCs increased.The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm.The ratiometric nanoprobes showed good selectivity for HPV-16 DNA,and the detection limit was 2 nmol/L.In addition,the practical applicability of this strategy was also demonstrated by analysing the HPV-16 DNA in human serum,illustrating its potential promise for clinical diagnosis.2 Ratiometric and sensitive detection of DNA based on chameleon fluorescent DNA-templated silver nanocluster and hairpin-blocked DNAzyme-assisted nucleic acid cascade amplificationThe previous work has realized the detection of HPV-16 DNA,but its sensitivity need be improved.On the basis of the previous work,we developed a simple and effective dual signal amplification method based on the combination of chameleon fluorescent DNA-templated silver nanocluster?DNA-AgNC?,hairpin-blocked DNAzyme probe?H-Dz?and catalytic hairpin assembly?CHA?to detect HPV-16DNA.In the absence of target,the H-Dz forms hairpin structure through intramolecular hybridization,which can inhibit the catalytic activity of DNAzyme.However,when the target exists,target-probe hybridization opens the hairpin and forms active secondary structure in the catalytic core to produce"act ive"DNAzyme,which cuts H-Dz with the help of Mg²⁺.Then,the shorter cut DNA fragment and the target are separated due to the unstable hybridization:the former triggers a downstream CHA reaction,which changes the fluorescent signal of DNA-AgNC from red to yellow;the latter hybridizes with another H-Dz to trigger next round of activation cycle to cut H-Dz.In this way,target recycling amplification resulted in significant signal changes,reaching a detection limit of 5.7 p mol/L.It is expected to provide a new platform for highly selective and sensitive analysis of HPV DNA.3 Chameleon fluorescent DNA-AgNC for simultaneous ratio detection of HPV-16 DNA and HPV-18 DNAIt is well known that HPV-16 and HPV-18 are the two main carcinogens,accounting for about 70%of all cervical tumors.Therefore,in this chapter,chameleon DNA-AgNC is used as a signal source to design three DNA probes,including the P1probe?targeting to HPV-18?,the P2 probe?targeting to HPV-16?,and the hairpin probe HP.First,P1 and P2 can hybridize with HP to form a complex to open the hairpin structure of HP.The fluorescence of DNA-AgNC is yellow at this time.When both targets are present,the target hybridizes with P1 and P2,respectively,to compete P1 and P2 from HP.At the same time,HP forms a hairpin structure due to intramolecular hybridization,which causes the fluorescence of DNA-AgNC to change from yellow to red,and realizes the simultaneous detection of HPV-16 and HPV-18.It can also be used for the detection of targets in serum samples.