Design of an electrochemical biosensor based on reduced graphene oxide conjugated with a pyrene-carbohydrate for the subfemtomolar detection of the cancer biomarker AMF/PGI

• Synthesis of a novel biosensor for the detection of the cancer biomarker AMF/PGI. • Rational design of the nanocomposite using reduced GO and a pyrene-sugar adduct. • Subfemtomolar detection limit achieved in both PBS and human plasma. • Design versatility for detecting other biomarkers via customized bioreceptors. Phosphoglucose isomerase (PGI) is a multifunctional protein involved in glucose metabolism that also functions extracellularly as a cytokine known as autocrine motility factor (AMF), promoting tumor cell migration, metastasis, and angiogenesis. Elevated levels of AMF/PGI in serum and urine are associated with cancer progression, making it a potential biomarker for early diagnosis and monitoring. However, current methods for the detection of AMF/PGI are limited by their complexity and insufficient sensitivity. In this work, we have developed a novel carbohydrate-based electrochemical biosensor using d -fructose 6-phosphate (F6P), one of the two substrates of PGI, as a biorecognition element for ultrasensitive AMF/PGI detection. The biosensor was constructed using an assembly of reduced graphene oxide (rGO) conjugated with a rationally designed, pyrene-based spacer that was covalently bonded to F6P and immobilized on a screen-printed carbon electrode (SPCE). The rGO/pyrene-F6P nanocomposites were synthetized through a chemical process and fully characterized using contact angle measurements, atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). These techniques were used to demonstrate the nanocomposite’s attachment to proteins. Using rabbit muscle PGI as a model of AMF, the biosensor exhibited high specificity and ultra-low limits of detection of 0.94 fM in Tris buffer and 0.79 fM in human plasma. This platform is a significant advancement in sugar-based electrochemical biosensing for protein biomarkers. Its modular design allows for the detection of other clinically relevant protein biomarkers by replacing F6P with alternative biorecognition elements.