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Establishing a stable, reliable and sensitive assay for the direct analysis in trace serum is of significance for the medical diagnosis and disease monitoring. We develop a MnO2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO2, AMAM) for the quantitative detection of alkaline phosphatase (ALP) in trace serum. Due to the physical isolation of MnO2 shell, AMAM is equipped with high stability, rarely happening the accidental aggregation. Particularly, the SERS signal is regulated by the etching of MnO2 shell, requiring not to change the local electromagnetic field or the molecular structure of Raman reporter (4-mercaptopyridine (Mpy)). Designating MnO2 shell as the Raman reference and the ratio of IMpy/IMnO2 as the response signal, AMAM system can obviously eliminate the experimental interference and achieve highly reliable SERS quantification for ALP. This ratiometric SERS system exhibits good reproducibility, high stability and sensitivity for the ALP analysis. AMAM system is able to perform the colorimetric and SERS dual-readout detection, which displays a wide linear dynamic range (0.1–70 U/L) for ALP with a detection limit of 0.079 U/L. Additionally, integrating with in-capillary SERS technique, our proposed nanoprobe is also applicable of direct SERS analysis in trace undiluted human serum (2 μL).

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.