Functionalized graphene oxide–antibody conjugate-based electrochemical immunosensors to detect Opisthorchis viverrini antigen in urine

Abstract

The liver fluke, Opisthorchis viverrini (OV), is the most important risk factor associated with cholangiocarcinoma (CCA) and its infection is a major public health burden in the Greater Mekong Subregion. The limitations of conventional diagnostic tools such as prolonged detection time and multistep procedures have led to an upsurge in demand for point-of-care testing that can facilitate timely and early action for better treatment. Herein, a novel, yet simple, electrochemical immunosensor for the onsite detection of OV antigen (Ag) in urine samples was developed. The modification of graphene-oxide (GO) surfaces with monoclonal anti-OV antibodies (mAb) through covalent conjugation not only shortened the time of antibody immobilization but also enhanced the sensitivity of the immunosensor. The successful conjugation of GO–mAb was characterized using functional and structural analysis. Furthermore, the electrochemical properties of the modified screen-printed sensor electrodes were verified by electrochemical and surface characterization. The developed electrochemical immunosensor achieved a low limit detection of OV Ag of 0.08 ng mL⁻¹ in standard buffer and 1.50 ng mL⁻¹ in urine. Moreover, it exhibited a broad linear range (0.2–200 μg mL⁻¹) of OV Ag detection and demonstrated excellent diagnostic potential with 93% sensitivity and 69% specificity compared to the traditional ELISA, and 92% sensitivity and 90% specificity compared to the commercial Rapid Diagnostic Test kit for urine OV Ag detection (OV-RDT). The highest AUC of 0.950 with 96% sensitivity and 90% specificity was obtained in comparison with the combined ELISA and RDT, without requiring the sample pre-treatment step. The results of the application of the developed immunosensor in the field-collected urine samples (n = 530) have further emphasized its potential utility as a prototype for point-of-care OV Ag detection.