▎ 摘　　要

Because of the complicated modification process, easy aggregation and low reproducibility, conventional Au-based SERS nanoprobes encountered a great bottleneck in clinical assay. We develop an alkyne functionalized graphene-isolated-Au-nanocrystal (AGIAN) SERS nanoprobe for the ratiometric analysis of alkaline phosphatase (ALP) in Raman-silent region with acetonitrile as the internal standard. Through a simple ultrasound treatment, the Raman reporter can be strongly bond on the graphene surface to prepare the AGIAN SERS nanoprobe within several minutes. Owing to the excellent physical isolation of graphene, the AGIAN nanoprobe possesses admirable stability, keeping off the irreversible metal aggregation. Moreover, with the intensity ratio between the Raman molecule (alkyne reporter) and the internal standard (acetonitrile solvent) as the signal, this nanoprobe is capable of reliable SERS detection of the ALP, effectively avoiding the optical fluctuations resulted from the external interferences. Particularly, the characterized peaks from the self-synthesized alkyne molecule and acetonitrile solvent are both strong and separated in Raman-silent region, suffering no signal overlapping with the biosystem and leading to reliable analytical results. Therefore, along with simple modification, high stability and accuracy, our SERS system provides a novel solution for the important appeal of SERS analysis in the clinical application.