Fluorescence immunoassay based on nitrogen doped carbon dots for the detection of human nuclear matrix protein NMP22 as biomarker for early stage diagnosis of bladder cancer

Abstract

A selective, fast acting and eco–friendly fluorescence immunoassay technique based on Nitrogen doped carbon dots (NCDs) is proposed for the detection of Nuclear Matrix Protein 22 (NMP22, antigen). Via the hydrothermal method and with the use of citric acid and urea, we synthesized NCDs with high quantum yield. Monoclonal antibodies (mAb, antibody) were labeled with NCDs through the EDC–NHS amidization technique and, in order to carry out the immunoreaction between the antigen and antibody, the resulting NCDs –mAb conjugates were then incubated with a small amount of NMP22). The established immunocomplex on the carboxylated NCDs led to the quenching of the fluorescence intensity. Under optimal conditions, a linear correlation between the change in fluorescence intensity and NMP22 concentration in the range of 1.3–16.3 ng/mL was observed and a detection limit of 0.047 ng/mL (47 pg/mL) was attained. This detection method was successfully able to determine NMP22 in human urine samples, with recoveries ranging from 96.50% to 103.61%. These results provide ample proof regarding the potential use of NCDs as fluorescence labels in immunoassays.