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 an aptamer-based biosensing strategy that overcomes this limitation by using selected single aptamer sequences for the precise and selective quantification of apo- and holo-RBP4 on graphene field-effect transistor (gFET) sensors. The specific contribution to this study is the identification of individual apo- and holo-RBP4-binding aptamers from previously enriched polyclonal libraries, their computational evaluation by molecular docking and molecular dynamics simulations, as well as their implementation as isoform-selective recognition elements on gFET sensors. The polyclonal aptamer libraries previously were generated using FluMag-SELEX (FluMag- Systematic Evolution of Ligands by Exponential Enrichment) that are 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.