Ultra-sensitive magnetic immunoassay of HE4 based on surface enhanced Raman spectroscopy

Human epididymis protein 4 (HE4), as a serological marker, has been proposed to be the most promising tumor marker in ovarian cancer diagnosis. An approach based on surface enhanced Raman spectroscopy (SERS) and a magnetic immunoassay technique was developed successfully for rapid detection and separation of HE4 with high sensitivity and selectivity. The detection was involved in the construction of a unique sandwich structure using a bottom-up method, which consisted of HE4 antibody and SERS reporter coated Au nanoparticles (A) and target HE4 antigen and HE4 antibody-modified magnetic core–shell Fe3O4@Au nanoparticles (B). The sandwich structure was effectively enriched by using a magnet for SERS detection. This approach exhibited an extremely high specificity in the detection of HE4 due to the strong specific interaction between the antibody and the corresponding antigen. The results revealed that the limit of detection (LOD) of the present approach was as low as 100 fg mL−1 and demonstrated a linear relationship between SERS intensities and lg c in a concentration range of 1 pg mL−1 to 10 ng mL−1. Accompanied by the magnetic enrichment procedure after the assembling of the sandwich structure, almost all of the HE4 protein was removed. The immuno Fe3O4@Au nanoparticles were regenerated by releasing the HE4 from the sandwich structure into the acidified methanol solution, and it could be used for magnetic enrichment and SERS detection for at least five times. Moreover, two kinds of immuno nanoparticles (A and B) could be developed as reagent kits in the clinical diagnosis of ovarian cancer.