Gold Nanoparticles For Imaging Of Cell Surface Specific Protein Glycosylation Based On Local Surface Resonance Effect

Protein glycosylation is one of the most important post-translational modifications in eukaryotes and plays a key role in living organisms.This unique modification not only affects the structure and function of the protein,but also plays a huge role in specific biological processes such as cell recognition,mucosal protection,material transport,cancer cell metastasis,and protein folding.Many studies have shown that in tumor cells,the glycosylation modification of certain glycoproteins will change with the occurrence and development of tumors,leading to abnormalities in their ligand binding,signal transduction,and molecular adhesion.Increased proliferation,apoptosis,invasion,and metastasis.Therefore,monitoring the dynamic changes of protein glycosylation has great research significance for the diagnosis and treatment of cancer.In recent years,based on a variety of imaging technologies,people have developed some detection methods suitable for protein-specific glycosylation levels.Currently widely used imaging methods are mainly based on nearby catalytic effects such as fluorescent resonance energy transfer(FRET)or surface-enhanced Raman spectroscopy(SERS).However,these methods also have certain shortcomings.In the imaging system based on FRET technology,because the current glycan labeling methods cannot achieve protein-specific labeling,the receptor fluorescent label is widely labeled on a certain sugar in the entire cell.On the model,a strong background fluorescence signal is generated,which reduces the signal-to-noise ratio of the imaging and the sensitivity is not strong;In addition,due to the limitations of the instrument technology,the SERS-based imaging system can only achieve cell-level detection and cannot be applied to the living body Imaging.Therefore,there is an urgent need to develop a method with high specificity,high sensitivity,and a wide range of applications for imaging of protein-specific glycosylation levels.At present,the gradual development of cross-fusion of nanotechnology and biomedicine has made great progress,bringing new development directions for protein-specific glycosylation imaging.Studies have shown that gold nanoparticles have unique optical properties,good biocompatibility,and surface modifiability,and have been widely used in many fields such as bioimaging,drug delivery,and nanotherapy.In addition,gold nanoparticles have a tunable localized surface plasmon resonance(LSPR)effect.When the particle size of the particles increases,the LSPR characteristic absorption peak will redshift and the light scattering intensity increases.At the same time,larger-sized gold nanoparticles can produce photoacoustic effects under the excitation of near-infrared light,and can be used as contrast agents for photoacoustic imaging.Therefore,gold nanoparticles are expected to become a powerful tool for protein-specific glycosylation imaging.In view of the importance of glycosylation analysis and the unique properties of gold nanoparticles,this article based on the local surface plasmon resonance effect of gold nanoparticles combined with DNA nanotechnology and sugar metabolism marker technology to achieve the specificity of MUC1 protein at the cell and living level Imaging of glycosylation level:1.Based on the resonance coupling effect of gold nanoparticles,a protein glycosylation analysis platform based on dark-field microscopy was developed to realize in situ analysis of sialic acid on the surface of MUC1 protein in living cells.The imaging system consists of two kinds of probes,a protein probe and a sugar probe.The protein probe is a functionalized aptamer S2.2 functionalized gold nanoparticle(30nm),which can target the MUC1 protein.The sugar probe is HS-PEG-DBCO-functionalized small-size gold nanoparticles(5nm),and sialic acid is labeled by a copper-free click reaction between DBCO and N3 group.Under dark-field microscopy,gold nanoparticles with a particle size of 30 nm appear as light green spots,while gold nanoparticles with a particle size of 5 nm cannot observe scattered light.When the two probes are bound to the target,a single-core-multisatellite plasma polymer is formed in situ at the target protein,and the LSPR effect is enhanced.Bright orange or even dark red spots can be observed by dark field microscope.The results show that this method is a fast and accurate research tool for protein glycosylation,and it can be more versatile with the changes of nucleic acid aptamers.2.Designed and developed a photoacoustic imaging platform based on hybrid chain reaction for quantitative imaging of specific glycosylation of MUC1 protein at the living level.The platform consists of a protein recognition probe,a sugar recognition probe,and a signal output probe.The protein recognition probe consists of three parts: the aptamer region that is responsible for identifying and targeting the binding protein,the HCR triggering chain region,and the spacer region.The sugar recognition probe is a DBCO-modified oligonucleotide,which is complementary to the structure of the spacer region,and is labeled on the glycan through sugar metabolism labeling and copper-free click chemistry.The signal output probe is a gold nanoparticle modified with a hairpin structure that participates in the hybrid chain reaction.When the sugar recognition probe and the spacer region are competitively hybridized,the hairpin structure is opened,the initiator chain region is exposed,the hybrid chain reaction with the signal output probe is triggered,and gold nanoparticles are generated in situ at the target protein.Aggregates can produce photoacoustic signals under the irradiation of 680 nm laser.The results show that this imaging method avoids the generation of background fluorescent signals by using DNA structure instead of fluorophores as markers of proteins and glycans,and successfully achieves MUC1 protein-specific glycosylation imaging at the living level.