The Catalytic Property Of Pt@mSiO₂ Nanozyme And Its Signal Amplification Application In Biological Detection

Enzyme-based signal amplification strategy is one of most popular techniques for biodetection. Although the enzyme has strong substrate specificity, high catalytic activity, low detection limit under mild reaction condition, the enzyme labeling process is always sophisticated and high cost. Additionally, enzyme also tends to denature under environmental changes such as pH value and temperature, which further leads to false positive results. Therefore, it is of great importance to construct the novel artificial enzyme-based signal amplification strategy. Since Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) were found to possess intrinsic peroxidase-like activity in 2007, the studies on the enzyme-like property of nanomaterial have been attracting more attentions. For instance, cerium oxide NPs, carbnon NPs, and noble metal NPs have been separately discovered to possess unique enzyme-mimic catalytic activities. Meanwhile, although the great progress was made over the above mentioned nanomaterials, in comparion with the natural enzymes, these nanomaterials as mimetic enzymes always possess poor catalytic performance and their catalytic properties could be greatly affected by the post surface modification, which further limits their application in bioassay. In order to overcome the above shortcomings, this paper focused on the platinum-based mimetic enzymes with the attention to further improve its catalytic performance by constructing mesoporous silica shell, and further expanded their application in biological detection. By modifying the surface of the as-synthesized platinum with silica coating, which effectively eliminates the influence from both ligand modification and biological molecules connection, both the catalytic activity and the dispersion stability have been greatly improved. Finally, the obtained nanocatalysts have been applied in single nucleotide polymorphisms (SNP) detection and enzyme linked immunosorbent assay (ELISA) as the enzyme substitute. The results were shown as follows:1. To screen nanozymes with the platinum and palladium as catalytic species and further study their catalytic properties.Based on the existing protocol for the synthesis of noble metal nanocatalyst, three kinds of nanozymes, i.e., Pd@Pt NPs, Pd@mSiO2@Pt NPs, Pt@mSiO2NPs, have been firstly prepared. And then their surfaces were further functionalized to facilitate the connection of biomolecule or keep the good dispersion. After screening the above nanozymes by systemically evaluating their performance in catalyzing the oxidation of TMB or the related dispersion stability, we found that Pt@mSiO2NPs could exhibit both excellent catalytic performance and good dispersion due to the protection of silica shell. All these results have laid a solid foundation for the following chapers where the Pt@mSiO2 nanozyme-based signal amplification strategies in biological detection were further studied.2. Studies on the catalytic performance of Pt@mSiO2 nanozyme and its application in SNP detection.Based on the above results in the second chapter, we further presented a novel label-free method for SNP detection by combining gated mSiCh and the catalytic signal amplification of Pt NPs, in which ssDNA caputured on the surface of Pt@mSiO2 could act as gate. Firstly, we have optimized the experimental conditions for the TMB-H2O2 colorimetric system, including pH, H2O2 concentration, TMB concentration, Pt@mSiO2 concentration. Steady-State Kinetics of Pt@mSiO2 was then studied using TMB-H2O2 as colorimetric model. And the results show that apparent Michaelis constant of substrate TMB and H2O2 (TMB Km= 2.35×10-8 mM, H2O2A:m=2.55×10-6mM) under this condition were lower than HRP (TMB Km= 0.434mM, H2O2 Km=3.70mM), indicating that the Pt@mSiO2has exhibited higher substrate affinity than that of HRP. After that, we further optimized the blocking conditions of ssDNA against Pt@mSiO2, such as the length of DNA, DNA concentration and incubation temperature. Finally, this system has been applied in the detection of single-base mutation associated with the breast cancer gene BRCA1 T0. The results show that when the incubation time is 3 min, the linear range of TO is 1-20 nM with detection limit of 3 nM. Meanwhile, Pt@mSiO2 has performed the good single base pair mismatch-discrimination capability and the relative absorbance ratios of T0/T1/T2/T3 achieved at 3 min is 1/0.55/0.47/0.4. Considering the fact that this method does not require labeling ssDNA probe in advance, it can be expected that it could have the good potential application value in clinical detection.3. The application of Pt@mSiO2 nanozyme in the ELISA as the enzyme substitute.In order to address the disadvantages of enzyme labeling in traditional ELISA, we attempted to replace the HRP with Pt@mSiO2 obtained above. We firstly prepared vatious CeO2 NPs, Fe3O4 NPs, Pt NPs according to the previous resport with the attention to use them as the contral. For the sake of comparing their capability in catalyzing in TMB substrate, the above prepared NPs were further modified. The results show that the surface modification could greatly inhibit their catalytic property. As a control, Pt@mSiO2 exhibits both high catalytic activity and good dispersion due to the protection of silica shell. In order to further improve its dispersion stability in PBS buffer, its surface was further modified with the spacer NHS-PEG-NHS, the resulting nanocomposite was further conjugated with secondary antibody Ab2. Finally, an artificial enzyme labeling assay for the dection of hCG has been established. The results show that after 5 min incubation there is a linear relationship between the absorbance and the logarithm of hCG concentration in the range of 5 to 200 ng/mL and LOD can reach 10 ng/mL.