New Methods Of Gene Editing And Biosensing Based On SWNTs-Based Novel Nanomaterials

In recent years,the concept of nanotechnology and its application in biomedicine have received extensive attention,combination nanotechnology and biotechnology have a bright future in biomedicine.The realization of nanotechnology is based on the development of nanomaterials,among them,carbon-based nanomaterials have attracted great interest in various research fields due to their unique structural dimensions and excellent physical and chemical properties.In this paper,based on carbon-based nanomaterials a new strategy in delivery and sensing has been developed.The specific designs are as follows:（1）In chapter 2,CRISPR/Cas9 as an efficient gene editing tool,how to safely and efficiently deliver it into cells and regulate it spatio-temporally remains challenging.With the development of nanotechnology,single-walled carbon nanotubes（SWNTs）have excellent physical and chemical properties and unique structural characteristics.Here we based on SWNTs designed a new method for photothermal activation of the CRISPR/Cas9 gene editing system.Firstly,branched polyethyleneimine（PEI）was modified by functional group cross-linking reaction,then the Cas9/sgRNA was connected by blocking DNA sequences which was stably hybridization with the target DNA binding regions in the sgRNA fragment.Finally,SWNT-PEI-DNA/sgRNA-Cas9（SW-PDsC）composite was synthesized.The SWNTs itself acts as a photothermal agents,can absorb light at 808 nm and convert it into heat,temperature rise cause DNA/sgRNA dissociate,thus release CRISPR-Cas9 on demand.The’proton sponge effect’of PEI can help materials escape from lysosomes,then the released sgRNA are led by the Cas9 protein nuclear localization signal（NLS）into the nucleus for gene editing.Intracellular delivery of CRISPR/Cas9 systems via exogenous control provides a new platform for targeted gene editing in deep tissues,aims to promote further development of delivery gene editing system in vivo by nanomaterial.（2）In chapter 3,nanozyme activity is an important property of nanomaterials,SWNTs possesses intrinsic peroxidase-like activity,however,there is an inherent defect of low catalytic activity.In order to excavate more potential of SWNTs in sensors,we attempted synthesis of SWNT-AuNPs complexes in situ to enhance the intrinsic peroxidase-like activity of SWNTs.PEI on the surface of SWNTs can provide a large amount of-NH₂,according to the interaction between-NH₂ and AuNPs,generated uniformly dispersed AuNPs in situ,the stability of AuNPs enhanced.Those nanocomposites in situ synthesized have better peroxidase activity than those by direct physical adsorption,in the presence of H₂O₂,successful detection of H₂O₂ and glucose concentrations using the typical TMB chromogenic assays.Provide a colorimetric detection platform for high-stability catalysis of artificial nanozymes based on carbon tubes.