Studies On Peroxidase-like Activity And Biological Applications Of DNA-based Metal Nanoclusters

DNA-templated metal nanoclusters have multiple enzymatic activities and show promising applications in bioanalysis,cell imaging and cancer therpy.In this paper,seven different sequences of single-stranded DNA,namely three C-rich sequences（kit,c-myc,HTC）containing different number of C bases,one G-rich sequence（TBA）and three CG-rich sequences（kit-TBA,c-myc-TBA,HTC-TBA）combining three C-rich sequences and TBA sequence,respectively,were designed as templates for the synthesis of monometallic nanoclusters（DNA-M NCs）,bimetallic nanoclusters（DNA-Fe/M NCs）and trimetallic nanoclusters（DNA-Fe/Cu/Ag NCs）.The nanoclusters were characterized by TEM and XPS.The peroxide-like activity of the nanoclusters and their influencing factors were mainly investigated,and the catalytic performance and catalytic mechanism were analyzed.Finally,the application in biological systems was achieved.The main research works in this paper are as follows:（1）Study on the peroxidase-like activity of DNA-templated metal nanoclusters.A series of kit-TBA-M NCs were synthesized using the kit-TBA sequence as template,and the peroxidase-like activities of these nanoclusters were tested using H₂O₂and TMB as substrates.It was found that the kit-TBA-Fe NCs had the highest peroxidase-like activities,and the effects of DNA template and the concentrations of metal ions on the peroxidase-like activities of nanoclusters were further investigated.The kit-TBA-Fe/Ag NCs,kit-TBA-Fe/Cu NCs,and kit-TBA-Fe/Cu/Ag NCs were also synthesized,and the temperature and p H of catalytic reactions were optimized.The catalytic activity was highest when the temperature of the reaction system was 25°C and the p H value was 4.0.Although the three nanocluster-like enzymatic activities were similar to that of the kit-TBA-Fe NCs,the kit-TBA-Fe/Ag NCs had slightly higher catalytic activity.The structure of the kit-TBA-Fe/Ag NCs and kit-TBA-Fe/Cu/Ag NCs was characterized by TEM and XPS,the average particle sizes were about 1.8±0.3 nm and 1.4±0.2 nm,and the main presence of metal elements in the nanoclusters in the form of Fe（Ⅱ）and Fe（III）,Ag（0）,Cu（0）and Cu（I）.（2）Study on the peroxidase-like activity of kit-TBA-templated metal nanoclusters.The four nanoclusters had good stability,and the relative activity decreased about 10%after 360 hours of placement.The catalytic activity could be improved by appropriately increasing the concentration of the nanoclusters,but too large concentrations instead reduced the catalytic activity.Steady-state kinetic experiments showed that the enzyme-like activity of kit-TBA-Fe/Ag NCs was slightly higher than that of other clusters because of the lower Michaelis constant（K_m）values for H₂O₂ and TMB,implies a stronger affinity for both substrates.Using terephthalic acid（TA）as a fluorescent probe,it was demonstrated that the hydroxyl radicals（·OH）were generated in the process of H₂O₂catalysis by nanoclusters.kit-TBA-Fe/Cu/Ag NCs showing the strongest fluorescence intensity,indicating that the synergy of multiple metal elements in the nanoclusters is more adequate to catalyze H₂O₂ by generating large amounts of·OH.（3）Application of kit-TBA-templated metal nanoclusters in biological systems.All four nanoclusters had inhibitory effects on the growth of cells,it was found kit-TBA-Fe/Cu/Ag NCs had the strongest cytotoxicity,catalyzing the production of large amounts of ROS from endogenous cellular H₂O₂,which could be seen as obvious green fluorescence after fluorescent dye treatment;the addition of exogenous H₂O₂ induced ROS production as well as altered cell morphology,resulting in enhanced toxicity.Perhaps because of the difference of endogenous H₂O₂ concentration in cancer cells and normal cells,kit-TBA-Fe/Cu/Ag NCs inhibited different inhibitory effects on cells,but both showed time and concentration dependence.The four nanoclusters showed good selectivity and anti-interference ability for the detection of GSH in buffer systems,with a linear range of 1.0-40μM,among which the kit-TBA-Fe/Ag NCs had the lowest detection limit of about 0.15μM for GSH.GSH detection in fetal bovine serum was achieved.