Hyaluronidase expression is known to be upregulated in various pathological conditions. A method based on a combination of ratiometric surface-enhanced Raman scattering (SERS) and magnetic separation is described for the determination of hyaluronidase (HAase) activity. Gold nanospheres (AuNPs) functionalized by 4-mercaptophenylboronic acid (4-MPBA) form stable cyclic esters with diol on hyaluronic acid (HA) by the boronic acid group, while Fe3O4@DTNB@Au modified with mercaptoethylamine (MEA) was used as a capture substrate to bind to the carboxyl group on the surface of HA, forming the "Au@4-MPBA@HA/Fe3O4@DTNB@Au@MEA" "core-satellite" structure. When HAase is present, HA is enzymatically disrupted, resulting in the destruction of the "core-satellite" structure, the SERS signal of 4-MPBA is subsequently weakened. The gold shell in the substrate protects the 5,5'-Dithio bis-(2-nitrobenzoic acid) (DTNB) from the external environment, which makes it become an ideal internal standard (IS) molecule for subsequent calibration. Under optimal conditions, the I1075/I1324 varied in the range of 10-3 - 10 U‧mL-1 HAase activity, with a limit of detection (LOD) of 0.32 mU‧mL-1，below the level of HAase in normal human body fluids. This method has been successfully applied to the determination of HAase activity in urine and is expected to provide a new method in disease detection, especially in the non-invasive detection of bladder cancer.
Keywords: Fe(3)O(4)@Au nanoparticles; Hyaluronidase (HAase); Ratiometric detection; Surface-enhanced Raman scattering (SERS).
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