The Application Of Conalent Noble Metal Graphite Nanoprobe In Biosensing

Biosensor is an emerging technology integrating biology,chemistry,physics,medicine and other disciplines.It is widely used in molecular detection,cancer treatment,cell imaging and so on.At present,sensors with high sensitivity and good cost performance are in increasing demand.Precious metals（Au,Ag）nanomaterials because of adjustable optical properties,excellent resistance to digestion ability and good biocompatibility and other advantages,has been widely used in biological sensing technology,precious metal materials surface assembly is an important process of constructing biological sensing system,it relates to the biological sensing system of sensitivity,selectivity and stability,etc.During the assembly process,the probe molecules containing sulfhydryl groups can be modified to the surface of gold or silver by either Au-S or Ag-S bonds.However,due to the weak force of Au-S bond or Ag-S bond,the mercapto probe molecules on the surface of the material are vulnerable to the competitive interference of biological mercaptan molecules and fall off,leading to false positive or false negative results.Looking for a more powerful covalent bond can solve the above problems.However,due to the lack of stable organic groups on the surface of traditional precious metals,it is difficult to provide a modification interface for other covalent bonding bonds.Graphene nanomaterials have good functionalized interfaces,stable chemical properties,excellent biocompatibility and optical properties,which can provide stable modification groups and functionalized interfaces for noble metal nanomaterials.In this paper,we combined the excellent properties of graphene nanomaterials and precious metal materials to construct covalent functionalized graphite-coated precious metal composite materials,and used it for intracellular molecular detection,cancer treatment and cell imaging.The main research contents are as follows:（1）Based on graphene-coated gold nanoparticles Au@Graphene（AuG）,a covalent system ofDNA and AuG NP,label-rcDNA-AuG,was prepared for intracellular miRNA-21 detection and cancer therapy.Firstly,N₃-NH₂ was modified onto the graphene surface of AuG NP by addition reaction,and NH₂-AuG nanoparticles with amino covalent functionalization were prepared.Furthermore,NH₂-AuG nanoparticles were covalently linked with carboxyl（COOH-）labeled capturedDNA（cDNA）to construct a covalently linked amide（-NH-CO-）cDNA-AuG system.Finally,a covalent nanoprobe label-rcDNA-AuG was prepared by the complementary pairing of labeledDNA（called label-rDNA）probe and cDNA base.In the absence of target,the fluorescence of the system is quenched by nanoparticles,and there is no toxicity.When the target miRNA-21（miR-21）is present,label-rDNA hybridizes with miR-21 to form a more stable double strand,which is released from the surface of AuG NP with enhanced fluorescence and toxicity,which can realize the detection of miR-21 and the treatment of cancer cells.The detection limit was 66 p M and the cancer cell mortality was 66.7%.In addition,the system has excellent anti-interference performance and stability,and can resist the interference of a variety of biological molecules（such as nuclease,DNA,glutathione,etc.）in cells.This strategy provides a new solution for monitoring intracellular molecules and tumor therapy.（2）Based on graphene-coated gold and silver alloy nanoparticles Au-Ag@Graphene（AuAgG）,Raman molecule and Au/Agg covalent nanoprobe were prepared for SERS imaging without interference in cells.First,amino-covalent functionalized nanoparticles NH₂-AuAgG were prepared by addition reaction on the graphene surface of AuAgG NPs.Then NH₂-AuAgG nanoparticles were covalently linked to Raman molecules with benzyl bromide or carboxyl group,and further covalently linked to carboxyl group（-COOH）on targeted polypeptide（MLS,TAT）.Four kinds of covalent Raman probes with targeted functions,AAGs-EA-MLS,AAGs-EA-TAT,AAGs-DA-MLS and AAGs-DA-TAT,were constructed for cell imaging.The strategy has good stability and excellent anti-interference ability.The new AuAgG nanocomposites can resist the corrosion of oxides and sulfides.Covalent bonding can avoid the interference of intracellular biological mercaptan.Raman molecules exited the peak in the silencing region of 1800-2400 cm^-1 cells to prevent the interference of other Raman peaks;Connect to targeted peptides for precise area imaging.This strategy provides a new approach for cell Raman imaging.