Detection Of Trace Copper With Methanobactin Functionalized Gold Nanoparticles

Traditional heavy metal ion detection methods are difficult to use for on-site detection,tedious and time-consuming.Gold nanoparticles?GNPs?and mimic enzymes are widely used in biochemical analysis because of their excellent properties.Methanobactin?Mb?is a copper-like peptide secreted extracellularly by methane-oxidizing bacteria during copper-limited culture.In this thesis,Mb-GNPs synthesized in the laboratory was used as tool.Based on the special surface plasmon resonance effect and high electron transfer ability of GNPs,the specific binding of Mb to Cu²⁺and the peroxidase simulation activity of Mb-Cu,a fast and trace copper detection method that can be used for on-site detection has been established.The main research contents are as follows:Mb with complete structure was obtained by copper-limited fermentation culture of Methylosinus trichosporium OB3b.Mb-GNPs were synthesized by Mb-indicated chloroauric acid reduction and the reaction system was optimized.Mb can specifically coordination bind to Cu²⁺through 4-sulfoyl-5-hydroxyimidazole?THI?and 4-hydroxy-5-thioimidazole?HTI?in the molecule.After adding the range of 1-5?mol/L Cu²⁺to Mb-GNPs solution,Mb-GNPs aggregated immediately.According to the surface plasmon resonance?SPR?effect of GNPs,the absorption peak of Mb-GNPs at 520 nm gradually decreased,a new characteristic peak was formed at 647 nm,accompanied by the color of solution change from red to blue-violet.It was found that the ratio of the absorption values of A647 to A520 was linear with the Cu²⁺concentration at range of 1-4?mol/L.Based on this,a visual detection method for Cu²⁺was established with the linear equations y=0.48x-0.39?R²=0.98?.The detection limit of the method was?3??0.34?M,and Co²⁺interfered slightly with the detection.With hydroquinone as substrate,the simulated peroxidase activity determination was established by the concentration change of hydroquinone before and after the reaction.The enhancement effect of Cu²⁺on the simulated peroxidase activity of Mb-GNPs was verified.A Cu²⁺-enhanced Mb-GNPs simulated peroxidase-catalyzed hydroquinone reaction system was constructed.The reaction system was optimized with Mb-GNPs particle size,reaction time,system pH,and temperature.It was found that there was a good linear relationship between the enzyme activity and Cu²⁺concentration at range of 10-600 nmol/L.Based on this,a Cu²⁺detection method was established with the linear equation y=0.025x+0.68?R²=0.989?.The detection limit of the method was?3??57.9 nM.The specificity analysis showed that this method had higher specificity and stronger anti-interference for Cu²⁺,and the detection limit and specificity were more advantageous than that of the visual detection in the previous chapter.The applicability of Cu²⁺detection in fresh bracken and celery with Mb-GNPs visual detection and simulated peroxidase detection was discussed.The results showed that both methods can be used to detect Cu²⁺in bracken and celery.The detection range of visual detection was 0.5-2.5?mol/L,the content of Cu²⁺in bracken and celery was 6.52 mg/L and13.3 mg/L,the spiked recovery ranges were 88.6%-104.5%and 91.5%-102%,respectively.The detection range of mimetic enzyme method was 0.2-1?mol/L,the content of Cu²⁺in bracken and celery was 8.6 mg/L and 10.4 mg/L,the spiked recoveries ranges were 85%-107.6%and 92.1%-106.8%,respectively.The copper content measured by both methods exceeded the national standard.The relative standard deviations of the two method groups are below 5%,which has good feasibility and precision.Both methods can be applied to detect Cu²⁺in real samples.