An epitope-imprinted electrochemical biosensor enabled by a thiolated graphene/AuNPs nanointerface for sensitive alpha-fetoprotein detection

A thiolated graphene/AuNPs nanointerface was constructed to enhance electron transfer. • An electropolymerized EMIP layer enabled selective epitope-based recognition of AFP. • A sensitive epitope-imprinted electrochemical biosensor was developed for AFP analysis. • The sensor exhibited a wide linear range (0.001–100.0 ng mL −1 ) and a low LOD of 0.069 pg mL −1 . • The biosensor enabled reliable AFP determination in spiked human plasma samples. Alpha-fetoprotein (AFP) is a clinically important serum biomarker for the early diagnosis of hepatocellular carcinoma. However, conventional AFP detection methods often suffer from limited sensitivity, selectivity and operational complexity. In this work, an electrochemical biosensor based on an epitope molecularly imprinted polymer (EMIP) was developed for the sensitive and selective determination of AFP. The sensing interface was constructed by electropolymerization of o -phenylenediamine ( o -PD) on a gold electrode sequentially modified with thiolated graphene (G–SH) and gold nanoparticles (AuNPs), using the AFP C-terminal dodecapeptide (KLISKTRAALGV) as the epitope template. The excellent analytical performance of the biosensor can be attributed to the synergistic integration of the EMIP recognition layer, G–SH, and AuNPs, which provide highly selective molecular recognition, an enlarged electroactive surface area and accelerated interfacial electron transfer. Under the optimized conditions, the proposed biosensor exhibited a wide linear range of 0.001–100.0 ng mL −1 , and a low detection limit of 0.069 pg mL −1 , together with excellent selectivity against NMP22, bovine serum albumin (BSA), human serum albumin (HSA), and neuron-specific enolase (NSE), good reproducibility, and satisfactory storage stability. Satisfactory recoveries obtained in spiked human plasma samples demonstrated its potential applicability for AFP determination in complex biological matrices. This work provides a sensitive and robust platform for AFP determination and highlights the potential of epitope-imprinted electrochemical biosensors for hepatocellular carcinoma screening and clinical biomarker monitoring. An epitope-imprinted electrochemical biosensor integrating an electropolymerized EMIP film with a thiolated graphene/AuNPs nanointerface was developed for AFP detection. The biosensor combines highly selective molecular recognition with enhanced electron transfer, achieving a 0.069 pg mL −1 detection limit and successful analysis of spiked human plasma samples. Download: Download high-res image (303KB) Download: Download full-size image