In this work, we developed a new type of SERS probe, which was composed of glass-SiO2-Au@MBN@Ag nanoparticles (NPs) three-dimensional Surface-enhanced Raman spectroscopy (SERS) substrate. When the laser passed through the quartz glass sheet, on the one hand, the SiO2 NPs supporting the Au@MBN@Ag NPs increase the roughness of the substrate surface, resulting in a large number of hot spots among nanoparticles. On the other hand, based on the focusing effect of silicon dioxide nanospheres, the laser can better focus on the surface of nanoparticles in the inverted SERS probe, thus showing better SERS enhancement. Furthermore, the Au@MBN@Ag NPs core-shell structure was used with 4-mercaptobenzoonitrile (MBN) as an internal standard molecule, and the quantitative determination of tyrosine and urea was realized by internal standard correction method. The standard working curves of the two had good linear correlation with R2 above 0.9555. The detection limits of tyrosine and urea were in the range of 2.85 × 10−10 M~7.54 × 10−6 M, which confirms that this design can be used for quantitative and specific detection of biological molecules, demonstrating great practical significance for the research of diseases such as skin lesions and endocrine disorders.
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