Challenges in Graphene-Based Biosensing: Exploring Critical Limitations and Strategies.

Pristine graphene, owing to its remarkable electrical and structural properties, has garnered significant interest as a sensing platform for such applications. In this study, we evaluate the use of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry to functionalize pristine graphene for the detection of antigens, essential for food safety and public health. However, our investigation reveals critical limitations in the reproducibility of both electrical and spectroscopic measurements post-functionalization. The unpredictable activation of pristine graphene surfaces via EDC-NHS coupling leads to inconsistent antigen immobilization, resulting in a high variability in sensor performance. Our studies, supported by Raman spectroscopy, indicate the absence of functionalization of the graphene surface via EDC-NHS chemistry. Our findings suggest that optimizing the functionalization protocol or employing alternative surface modification techniques, such as defect engineering or using linker molecules, is necessary to achieve consistent and reliable detection. This study sheds light on the challenges of using EDC-NHS chemistry with pristine graphene and provides a roadmap for improving sensor reproducibility in the detection of penicillin and cephalexin.