Research On Detection Of Some Disease Related Genes Based On The Fluorescence DNA-templated Silver Nanoclusters

The nucleic acids aptamer has been applied widely for the construction of biosensor probe because of these advantages of high affinity and specificity,small molecular weight,low immunogenicity,high thermal /chemical stability,wide recognition range of target molecules,simple operation and low cost and so on.In recent years,the Ag NCs synthesized using DNA as the template(DNA-Ag NCs)as a novel fluorophore present many unique physical and chemical properties,which attribute to its quantum effects,small size effects and surface effects and so on.So,insteading of traditional fluorescent markers,DNA-Ag NCs are extensively applied in the fields of biosensing and bioimaging.Based on these facts above,we develop a fluorescent DNA-Ag NCs nucleic acid probe for biosensing detection of certain disease related genes.The designed probe contains three domains: the C-rich sequence for synthesis of Ag NCs,the aptamer sequence that is fully complementary to the target DNA and the blocking sequence that is partially complementary to the C-rich sequence.When is annealed in PBS buffer,the random probe in the normal state will self-assemble into the locked hairpin structure as a result of the base pairs between the Ag NCs synthetic sequence(5?-terminus)and the blocking segment(3?-terminus).In the absence of synthetic template,the Ag atoms are particularly easy to aggregate irreversibly to form larger nanoparticles without fluorescence properties.On the contrary,if the target is introduced at this time,the C-rich DNA sequence will be freed from locked patterns due to the complete complementary pairing between the target and the recognition sequence on the probe.Ultimately,it is the strong fluorescence of the as-synthesized DNA-Ag NCs provides output signal and thus the detection of the target is successfully realized.By embedding different recognition sequences,the molecular beacon can also be conveniently used for versatile detection of diverse targets.Furthermore,if the emission spectra of these as-synthesized Ag NCs do not overlap,the constructed probes present a great potential in simultaneous detection of the multiple target genes.The main contents are as follows:(1)Optical properties of DNA-Ag NCs are highly dependent on the sequence,length and secondary structure of DNA template,which is the reason why we select four common sequences C-rich ssDNA squences in the literature as templates to design four different probes for HIV gene detection.Results indicate that the fluorescent enhancement of the probe 4 shows a significant superiority over that of HIV probes 1,2,and 3 upon associating with the target HIV.When the target HIV is added,the fluorescence intensity of system based on HIV probe 4 increases almost 20-fold.Inspired by the design of the HIV probe 4,the other two detection probes are also constructed by just replacing corresponding aptamer sequence and give the positive responses for the corresponding target H1N1 and H5N1.Among them the H1N1 probe presents the best biosensor result.Comparing with H1N1 probe alone,the fluorescence intensity of the system containing both the probe and target increases dramatically by 400 times.While the fluorescence intensity increases about 10-fold for the system containing the H5N1 probe and target compared with the H5N1 probe alone.(2)The feasibility of the detection mechanism is verified in PBS buffer by UV-Vis absorption spectrum,Fluorescence spectrum,Transmission electron microscopy,Gel electrophoresis analysis and Circular dichroism etc..Based on the optimized experimental conditions,three infectious disease-related genes HIV,H1N1,and H5N1 are detected,and the corresponding detection limits are 3.53,0.12 and 3.95 nM,the linear range are 5-2000 nM、10-250 nM and 50-500 nM,respectively.In addition,we also carried out the reproducibility experiment of target in the diluted fetal bovine serum to explore the application prospect of the system.(3)We are delighted to find that the constructed detection system allows simultaneous detection of HIV,H1N1,H5N1 genes,which is attributed to non-overlapping emission spectra of the as-synthesized silver nanoclusters.(4)Based on the sequence design of HIV probe,the p53 gene detection system is constructed and gives a negative fluorescence response.In this work,three common C-rich ssDNA sequences are selected for the design of probe.By comparing the fluorescence response of target to the six probes,the p53 probe 1 is selected for the quantitative detection of p53 gene based on the same sensing mechanism,the detection limits is 3.57 nM and the linear range is 250-2500 nM.