Impedimetric Graphene-BasedGold Immunosensor forColorectal Cancer Biomarker CCSP‑2 Detection

Abstract

Electrochemical immunosensors are emerging as promising tools for cancer detection due to their simplicity, portability, and high sensitivity. Colorectal cancer (CRC), the third most common cancer in the United States, remains challenging to diagnose early, as the standard method, colonoscopy, is invasive and often avoided. To address this gap, a graphene-gold-based immunosensor was developed for the early detection of CRC by targeting colon cancer-secreted protein-2 (CCSP-2), a biomarker overexpressed in CRC patients. The sensor was fabricated by attaching graphene oxide (GO) to a gold (Au) electrode using 4-aminothiophenol (4-ATP) as a linker, followed by immobilization of CCSP-2 antibodies (Anti-CCSP-2) and blocking with bovine serum albumin (BSA). Characterization of the immunosensor using linear sweep voltammetry (LSV), Raman spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) confirmed successful modifications. The attachment of graphene oxide contributed to an enhancement in current response, likely due to partial reduction and improved electron transfer at the modified surface. The sensor demonstrated a good linear response (R² = 0.979) to CCSP-2 antigen (CCSP-2) concentrations ranging from 1 ng/μL to 100 ng/μL, with a limit of detection (LOD) of 0.17 ng/μL and a sensitivity of 0.031 (ng/μL)⁻¹. Selectivity was validated using CRC cell extracts (CACO-2) and human kidney extracts (HEK), showing a more significant signal for CACO-2. These findings suggest that the developed immunosensor is a reliable and sensitive platform for CCSP-2 detection, with the potential for adaptation as a point-of-care device for early CRC screening.