Development of dual-mode ELISA based on ALP-catalyzed APP hydrolysis for IL-6 detection

Sensitive and accurate detection of interleukin 6 (IL-6) is crucial for the early diagnosis of cerebral infarction to improve patient survival rates. However, the low-abundance of IL-6 in cerebral infarction presents a significant challenge in developing effective diagnosis method. Herein, we studied and analyzed the strong fluorescence property of 4-aminophenol phosphate (APP) and developed an enzyme-linked immunosorbent assay (ELISA) for IL-6 detection. The detection was based on the integration of optical signal change induced by alkaline phosphatase (ALP)-catalyzed APP hydrolysis and ALP-mediated ELISA. The generated colorimetric signal of 4-aminophenol, APP hydrolysis product, was used for ELISA of IL-6 with a detection limit of 0.1 ng/mL, and the visual detection of IL-6 was achieved. The changes in APP fluorescence have a good linear relationship with the logarithm of IL-6 concentration in the range of 0.005 ng/mL to 5.0 ng/mL, with a detection limit of 0.001 ng/mL, which was 100 times lower than that of conventional pNPP-based ELISA. Furthermore, the constructed ELISA effectively distinguished between samples from patients with cerebral infarction and volunteers with non-cerebral infarction, and the severity of symptoms was well distinguished based on IL-6 measurement. The dual-mode ELISA demonstrated high feasibility of low-abundance biomarker detection and displayed good potential for accurate in vitro diagnosis.