Single-sequence based gFET-aptasensors for the discrimination of apo- and holo-RBP4 in human serum

Retinol-binding protein 4 (RBP4) transports vitamin A in blood as holo-RBP4 or apo-RBP4, and imbalances between these forms are linked to metabolic and cardiovascular diseases. However, conventional methods such as ELISA and Western blot cannot distinguish between them. Here, we present an aptamer-based graphene field-effect transistor (gFET) biosensor enabling selective quantification of apo- and holo-RBP4. Conformer-specific polyclonal aptamer libraries were generated by FluMag-SELEX, and next-generation sequencing with bioinformatic analysis identified two high-affinity aptamers. Computational modelling revealed their conformer-specific binding mechanisms. Immobilized on gFETs, these aptamers enabled label-free, real-time detection with picomolar sensitivity, representing a 100-fold improvement over the original libraries, with further gains achieved by increasing aptamer density. This work highlights aptamers as powerful biorecognition elements for precise RBP4 isoform monitoring.