Synthesis Of Oligonucleotide-capped Etal Nanoparticles And The Application For Biothiols Detection

Biothiols involve in the protein synthesis, metabolism and detoxcification, the unbalanced quantity level of Biothiols may induce diseases such as cardiac-cerebral vascular disease and Alzheimer’s disease. Therefore, it is necessary to monitor the content of biothiols inside organisms. Based on the reactivity of thiol groups with metal ions, nanoclusters and nanocrystals, many detection methods have been found currently, such as Electrochemical method, Electrochemical luminescence method,Fluorescence analysis method and Colorimetric method etc. In colorimetric method,color of products is employed as a factor to measure or compare the color of the solution contained colored substances. This is a really simple method, and the color is obvious, so it is usually adopted as a quantitive analysis tool in environment, food science and biomedical fields. However, to achieve a high sensitivity in colorimetric detection is still a challenge. In this paper, we use cytosine-rich oligonucleotide as a template to synthesize bimetallic nanoparticles Au_xPt_y, which are employed to explore supersensitive assays of biothiols.Firstly, cytosine-rich oligonucleotide was used for template to synthesize metal nanoparticles, Au, Pt and Au_xPt_y in experiments, and High-resolution Transmission electron microscopy（TEM）, Circular Dichroism Chiroptical Spectrometer（CD）,Ultraviolet visible spectrophotometer（UV-vis）, Fluorescence spectrophotometer（FL）and X-ray photoelectron spectrometer（XPS） were used to characterize nanoparticles.Results show the sizes of nanoparticles are: 4.8 nm-Au, 4.2 nm-Pt, 2.6 nm-Au₁Pt₁,and 3.6 nm-Au₂Pt₁ respectively. Au³⁺ is almost all reduced to Au0, Pt²⁺ is partly reduced to Pt0, Pt0/Pt²⁺ in Au₂Pt₁ is higher than in Pt and Au₁Pt₁, which indirectly shows that Au³⁺ is easy to be reduced than Pt²⁺, Au³⁺-coordination with the bases of nucleic acids decreases the proportion of DNA-bound Pt²⁺. From the results of UV-vis and CD, it can be deduced that Au and Pt coexist on the C-rich template in bimetal Au_xPt_y nanoparticles.Next, we evaluated catalytic properties of the metal nanoparticles in TMB-H₂O₂ reactions. The order of initial catalytic reaction rate is: Pt > Au₁Pt₁ >Au₂Pt₁, and reaction reaches equilibrium within ten minutes when catalyzing theoxidation of TMB with 700 nM Au₂Pt₁. This shows Au₂Pt₁ nanoparticle has high peroxidase activity, the kinetic parameters including Km and Vmax are determined via Michaelis Menten-double reciprocal plot, although the affinity of Au₂Pt₁ to 3, 3’, 5,5’-tetramethyl benzidine（TMB） is less than natural Horse Radish Peroxidase（HRP）,the maximum reaction rate is faster than HRP in TMB- H₂O₂ reaction.Finally, by comparing the lowest detection limit（LOD） to biothiols of Pt, Au_xPt_y,we learn that bimetal nanoparticles Au₂Pt₁ is best, its LOD for Cys, Hcy is 3.5 nM and 1.6 nM, respectively, compared with other colorimetry in table 2.2, the LOD of this method is lowest. We also found thiol detection performance of metal nanoparticles Au_xPt_y is related to the Au: Pt ratio（x:y） by using Isothermal calorimetry titration（ITC）, HR-TEM and XPS. In addition, the selective experiments show it is specific to biothiols detection.Due to the interaction between bimetallic nanoparticles Au₂Pt₁ and sulfhydryl compounds, Au-S bond is formed and nanoparticles aggregate gradually. The aggregations lower catalytic activity of metal nano-enzymes in TMB-H₂O₂ reaction.Therefore, according to the inhibition effect of Cys on Au₂Pt₁ in catalyzing TMB-H₂O₂, combining with high specificity of bimetallic nanoparticles Au₂Pt₁ to biothiols detection, Au₂Pt₁ was applied to detect biothiols in human serum. Ultimately,the concentration of 312 μM in human serum was achieved, this value is consistent with the literature. Following recovery rate has proved the reliability of this detection method in adding recovery experiments. In future, further study on bimetallic nanoparticles is expected to detect thiol-containing medicines.