Alkaline phosphatase (ALP) is a biomarker for many diseases, and monitoring its activity level is important for disease diagnosis and treatment. In this study, we used the microdroplet technology combined with an in situ laser-induced polymerization method to prepare the Ag nanoparticle (AgNP) doped hydrogel microbeads (HMBs) with adjustable pore sizes that allow small molecules to enter while blocking large molecules. The AgNPs embedded in the hydrogel microspheres can provide SERS activity, improving the SERS signal of small molecules that diffuse to the AgNPs. A specific hydrolysis reaction of ALP on 5-bromo-4-chloro-3-indolylphosphate (BCIP) was introduced and itsproduct 5,5'-dibromo-4,4'-dichloro-1H,1H-[2,2']bisindolyl-3,3'-dione (BCI) was employed to assess ALP activity due to its highly resonance Raman activity. The sensing platform was applied to model ALP activity in serum and evaluate ALP inhibitors. The SERS assay showed higher sensitivity than UV-vis absorption spectroscopy, with the lowest detectable ALP concentration of 1.0 × 10-20 M. In addition, the ALP activity in HepG2 cells was evaluated using this sensing platform, showing lower ALP-expressing activity than that of controls in response to hypoxia and iron metastasis. This SERS-activated HMB shows great potential in detecting ALP and is expected to help analyze complex clinical samples.
Keywords: SERS substrate; alkaline phosphatase; hydrogel microspheres; microfluidics; tunable pore size.