Development of nanosensor by bioorthogonal reaction for multi-detection of the biomarkers of hepatocellular carcinoma

A sensitive and specific multiplex immunoassay for the biomarkers of hepatocellular carcinoma (HCC), Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73), has been developed. The enzymes, Horse radish peroxidase (HRP) and Alkaline phosphatase (ALP) were modified with active reactive groups of click chemistry, and then self-assembled to form enzyme aggregates, HRPn and ALPn, respectively. The carbon nanotubes (CNT) were conjugated with the enzyme aggregates and the detection antibody of biomarkers by bioorthogonal reaction, forming the immuno nanosensor for AFP and GP73 individually. The enzymes on the nanosensor can catalyze its substrate to emit chemiluminescences, which can be read out in different time windows. Owing to the high reaction efficiency of the bioorthogonal reaction, large capacity of the catalytic enzymes has been conjugated to the surface of CNT, and then the signal intensity of chemiluminiscence has been greatly amplified. The multiplex immunoassay has been successfully applied to detect the biomarkers in real serum samples, which can improve the accuracy and preciseness for the clinical diagnosis of HCC. The nanosensors have high specifity for the biomarkers of HCC, and their sensitivities can be adjustable according to practical demand. This work provides a novel clue to develope immunoassay for clinical diagnosis of HCC.