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

Retinol-binding protein 4 (RBP4) is a key transporter of all-trans-retinol (vitamin A), circulating in blood as either holo-RBP4 (retinol-bound) or apo-RBP4 (retinol-free). Dysregulated RBP4 levels, particularly an imbalance between apo- and holo-RBP4, have been implicated in a range of metabolic and cardiovascular diseases. Current detection methods such as ELISA and Western blot lack the specificity to distinguish these two forms. Here, we present a novel aptamer-based biosensing strategy that overcomes this limitation, enabling the precise and selective quantification of apo- and holo-RBP4 using graphene field-effect transistor (gFET) sensors. Using FluMag-SELEX, we previously generated polyclonal aptamer libraries specific to each RBP4 conformer. Next-generation sequencing and bioinformatic enrichment analyses identified two highly specific and high-affinity aptamers, which were further characterized via computational modelling, including molecular docking and molecular dynamics simulations. This structural approach elucidated the conformer-specific binding mechanisms, demonstrating aptamers' capacity to differentiate subtle protein conformational changes. The selected aptamers were immobilized on gFET devices, enabling label-free, real-time detection with picomolar sensitivity – a 100-fold improvement over the original libraries. Increased aptamer density on the sensor surface further enhanced signal sensitivity by an order of magnitude. These findings validate the use of aptamers as high-performance biorecognition elements in biosensors, offering a path toward precise RBP4 isoform monitoring.