Construction Of DNAzyme Nano-Motor For Imaging Myeloperoxidase In Living Cells

Objective:Most inflammatory reactions involve the release of myeloperoxidase（MPO）.The expression level of MPO in tissue is associated with many diseases,such as cardiovascular system diseases and nervous system diseases.MPO has been used as a biomarker for a variety of diseases.The analysis and detection of MPO are of great significance in the diagnosis,treatment and prognosis of diseases.The existing methods have some limitations,so it is necessary to develop real-time and fast response probes for MPO detection.By connecting the DNAzyme chain and substrate chain on the gold nanoparticles（Au NPs）surface,a novel DNAzyme nanomotor was designed and developed to achieve high-sensitivity detection of hypochlorous acid（HCl O）and MPO.In addition,based on its superior biocompatibility and stability,MPO in living cells can be detected by fluorescence imaging strategy at the same time,providing new strategies and ideas for the evaluation of MPO as a marker of diseases.Methods:The DNAzyme motor was constructed using Au NPs as carriers and the DNAzyme chain and substrate chain were loaded on its surface using the salt aging method.UV-vis was used to detect the change in the absorption spectrum of the DNAzyme motor.The changes in particle size and Zeta potential were detected by Zeta potential analyzer.The fluorescence release of the DNAzyme motor was detected by a fluorescence kinetic experiment.Cell survival rate was detected by the CCK8 method.DNAzyme motor cell intake was detected by ICP-MS.Recognition and imaging of HCl O and MPO in living cells by DNAzyme motor under confocal laser fluorescence microscopy.Results:1.Construction and optimization of DNAzyme motor After the DNA strand was connected to Au NPs,the peak of the UV-visible absorption spectrum showed a redshift phenomenon,the particle size increased,and the negative Zeta potential decreased.These data indicated that the DNA strand was successfully connected to Au NPs.It is determined that the substrate chain load can be stabilized at more than400,which proves that the DNAzyme motor can be stably synthesized.PAGE experiment shows the feasibility of the design of the DNAzyme chain to recognize hypochlorous acid.The kinetic experiment successfully verified that the fluorescence intensity gradually increased with the increase of concentration and time,indicating that the DNAzyme motor can be used for the detection of hypochlorous acid and MPO.In order to optimize the experimental conditions,100 m M Na Cl and 25 m M Tris-CH₃COOH with a Ph of 8 were selected as the reaction solution.By comparing different metal ions,Mn²⁺was selected as the cofactor.The concentration of Mn²⁺was optimized,and 0.75 m M was selected as the final concentration for the reaction.The ratio of Au NPs:DNAzyme:substrate was optimized,and a ratio of 1:200:1200was selected to construct the DNAzyme motor.According to the results of DNAzyme motors constructed with different DNAzyme chains,MPO-1 was selected as the enzyme chain for the construction of DNAzyme motors in this study.2.Stability and repeatability of DNAzyme nanomotors The influence of12h FBS and GSH on the stability of the DNAzyme motor was continuously tested.The results showed that there was no release of fluorescence within 12 h,and fluorescence only appeared after the addition of mercaptoethanol.At the same time,different batches of synthetic DNAzyme motors were tested,and the results showed that in the presence of the same concentration of hypochlorous acid,the motors could all move,and there was no significant difference between them.3.In-tube application of DNAzyme nanomotor The results of the detection of hypochlorous acid by DNAzyme motor showed that the fluorescence intensity increased with the increase of hypochlorous acid concentration,and had a good linear relationship in the range of 0.1～1μM.The detection limit of hypochlorous acid was finally calculated to be 13 n M.The results of MPO detection showed that the fluorescence intensity increased with the increase of MPO concentration,and had a good linear relationship in the concentration range of 0.6～7μg/m L,with the lowest detection limit of 38 ng/m L.4.Intracellular applications of DNAzyme nanomotors The results of CCK8showed that 0～1 n M DNAzyme motor had no toxic effect on cells for 24 h.The results of ICP-MS showed that the cell intake of DNAzyme motor increased with the increase of concentration and time.Laser confocal fluorescence microscopy observed that the DNAzyme motor can be used for exogenous hypochlorite imaging and endogenous MPO imaging.Conclusions:1.A novel DNAzyme nanomotor was constructed by mounting DNAzyme chain and substrate chain on Au NPs surface.2.The DNAzyme motor has the function of fluorescence amplification.3.The DNAzyme motor has unique properties such as fast response and good biocompatibility,showing excellent performance in hypochlorite and MPO detection.4.The DNAzyme motor has been successfully used for biological imaging of exogenous hypochlorous acid and endogenous MPO in living cells.