Preparation Of Ordered Surface-enhanced Raman Substrates And Their Application In Exosome Detection

Exosomes,extracellular vesicles with double membrane structure,are actively secreted by a broad range of cell types.They are rich in a large number of biomolecules,such as proteins,lipids and nucleic acids.The key function of exosomes is to mediate the communication between adjacent and remote cells,and to participate in the regulation of recipient cells by transporting related biomolecules to recipient cells,thereby affecting the biological functions of recipient cells.For example,micro RNA(mi RNA)molecules present in the lumen of exosomes secreted by the donor cells can be transferred to the recipient cells through binding between the exosomes and the recipient cells.This delivery can bypass the transcriptional control of recipient cells and directly mediate the regulation of gene expression,which is closely related to many physiological and pathological phenomena.Therefore,exosomes and their biomolecules have become biomarkers for the accurate diagnosis and treatment of related diseases.The detection and analysis of exosomes and their biomolecules are of great significance in the early clinical diagnosis and treatment of diseases.Surface-enhanced Raman spectroscopy(SERS)technology not only has extremely high detection sensitivity and spectral resolution,but also can provide detailed structural information of analyte molecules,which has become an important tool for biosensor analysis.Therefore,this paper developed new methods based on SERS technology for the highly sensitive detection of exosomes and exosomal mi RNA:1.Based on Au nanoctahedra array as SERS detection platform,we developed an accurate quantitative detection method for mi RNA let-7a in exosomes secreted by breast cancer cells MCF-7.We first synthesized Au octahedral nanoparticles,and self-assembled them into ordered monolayer film with large area through the method of self-assembly at the liquid-liquid interface.All the Au nanoctahedra in the film could uniformly"stand"on their triangle face.This kind orientation formed a highly uniform Au nanoctahedra array,and could provide a uniformly distributed"hot surface"for SERS detection,thus greatly improving the detection sensitivity and uniformity.The experimental results showed that the SERS enhanced activity of the Au nanoctahedra array was high(the enhancement factor was 10⁸),and the signal reproducibility was great(the relative standard deviation was 4.81%).Based on the array as the detection platform,we developed a highly sensitive SERS detection method for let-7a,which shows a broad linear range(10 amol/L to 10nmol/L)and a low detection limit(5.3 amol/L)without using any signal amplification strategy,and has high detection specificity,which can identify single-base mismatch.We used this method for the detection of let-7a in MCF-7 cells-derived exosomes.The results showed that the expression of let-7a in MCF-7 exosomes was only 0.01 copies,which was consistent with the results obtained by q RT-PCR method,indicating that the developed method was correct and reliable.We further evaluated the effect of drug treatment on the expression level of exosomal let-7a using this method.These studies indicate that this method has a potential application in clinical diagnosis and treatment.2.We developed a direct SERS detection method for exosomes based on gold nanobowls array.Ordered gold nanobowls array was prepared by template method.First,we synthesized the Si O₂ spheres,and assembled them on the flat copper wafer in a hexagonal close-packed manner by means of covalent bonding and self-assembly technology.Then,we evaporated the gold film on the Si O₂ template,and finally obtained the gold nanobowls array by etching method.By controlling the size of the Si O₂ spheres,the thickness of the gold film and the etching time,the size of the gold nanobowls was adjusted to match the size of the exosomes(?100 nm),so that exosomes could be captured into the nanobowls.Taking the gold nanobowls array as the SERS detection platform,we established a direct SERS detection method for exosomes secreted by MCF-7 cells,indicating the biosensing potential of this approach.