| Objective: Based on strand displacement-driven DNA self-assembly and catalytic nucleic acid,we attempted to establish a new biosensing system with simplicity,rapidity,sensitivity and specificity for detecting microRNA-122-5p(miR-122-5p)and further to evaluate the analytical performance of the detection system,thus providing a new strategy for the detection of miR-122-5p.Methods: 1.Establishment of detection system:(1)three hairpin probes Hairpin1(HP1),Hairpin 2(HP2),and Hairpin 3(HP3)were designed according to miR-122-5p,respectively.MiR-122-5p initiate strand displacement reaction,and then drive HP1?HP2 and HP3 to assemble DNA trigeminal structure compatible with the G-rich sequence.Whether the DNA trigeminal structure was successfully constructed was confirmed by agarose gel electrophoresis and atomic force microscopy from molecular weight and morphology,respectively.(2)The G-rich sequence forms a G-quadruplex after the chelation of hemin.A chemiluminescent sensing platform and a fluorescence sensing platform were constructed by the characteristics of G-quadruplex with horseradish peroxidase mimicking enzyme activity and the quenching effect.The feasibility of the two platforms was verified by comparing the chemiluminescence signal intensity and fluorescence signal intensity between the experimental group(with miR-122-5p)and the control group(without miR-122-5p).Optimization of key experimental conditions for chemiluminescence and fluorescence sensing platforms was based on orthogonal experiments(reaction time,reaction temperature,the concentration of Hemin?HP1/HC1(-CY3)?HP2/HC2(-CY3)and HP3/HC3(-CY3),et al.).2.Evaluation of the detection system: Compare the sensitivity of the chemiluminescence sensing platform and the fluorescence sensing platform,and then select a sensing platform with higher sensitivity to further evaluate its repeatability,specificity,matrix effect and stability.Results: 1.Establishment of detection system:(1)Agarose gel electrophoresis results show that the miR-122-5p+HP1+HP2+HP3 in lane has a 150 bp electrophoresis band,while the HP1+HP2+HP3 lane has a size of 50 bp electrophoresis band.Moreover,the atomic force microscope can visually verify the formation of a DNA trigeminal structure of about 20 nm in size.(2)Feasibility analysis of the chemiluminescence sensing platform showed that the luminescence intensity with miR-122-5p was 12369.17±1120.37 a.?.,and was 3098.01±121.35 a.?.without miR-122-5p,and the difference was statistically significant(P < 0.01).The results of orthogonal experiments showed that the optimal reaction time,reaction temperature,Hemin,HP1,HP2 and HP3 concentrations were at 90 min,42?,100 nM,150 nM,200 nM and 100 nM,respectively.The feasibility analysis of the fluorescence sensing platform showed that the fluorescence intensity with miR-122-5p was 85.19±5.36 a.?.,and was 213.7±14.99 a.?.without miR-122-5p,and the difference was statistically significant(P<0.01).The results of orthogonal experiments showed that the optimal reaction time,reaction temperature,Hemin,HP1,HP2 and HP3 concentrations were at 60 min,42?,100 nM,150 nM,200 nM and 100 nM,respectively.2.Evaluation of the detection system: sensitivity analysis shows that the minimum detection limit of the chemiluminescence sensing platform is 1.02 pM,and the fluorescence sensing platform is 5.70 fM.Repeatability experiments showed that the relative standard deviation(RSD)of different concentrations of miR-122-5p was less than 7%.Specificity analysis showed that the fluorescence intensity of single-base mismatched sequence,double-base mismatched sequence,four-base mismatched sequence and miR-21 was higher than that of miR-122-5p,and the difference was statistically significant(P<0.01).The recovery analysis showed that the matrix effect of different concentrations of miR-122-5p were lower than 6.5%.The stability analysis showed that there was no significant difference in fluorescence intensity values measured at 1 day,2 days,3 days,4 days,and 5 days,(P>0.05).Conclusion: This experiment successfully constructed DNA trigeminic nanostructures based on DNA strand displacement reaction and achieved sensitive detection of miR-122-5p due to the signal amplification function of the DNA assembly and catalytic nucleic acid(G-quadruplex).The sensitivity of the fluorescence sensing platform is higher than that of the chemiluminescent sensing platform with a low detection limit of 5.70 fM.Moreover,the repeatability,recovery,stability are domancted with good preformcnance and the specificity is at single base mismatched discrimination,which provide a new sensing detection strategy for the detection of miR-122-5p. |
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