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A label-free biosensor is developed for the determination of plasma-based Aβ 1–42  biomarker in Alzheimer’s disease (AD). The platform is based on highly conductive dual-layer of graphene and electrochemically reduced graphene oxide (rGO). The modification of dual-layer with 1-pyrenebutyric acid  N -hydroxysuccinimide ester (Pyr-NHS) is achieved to facilitate immobilization of H31L21 antibody. The effect of these modifications were studied with morphological, spectral and electrochemical techniques. The response of the biosensor was evaluated using differential pulse voltammetry (DPV). The data was acquired at a working potential of ~ 180 mV and a scan rate of 50 mV s −1 . A low limit of detection (LOD) of 2.398 pM is achieved over a wide linear range from 11 pM to 55 nM. The biosensor exhibits excellent specificity over Aβ 1–40  and ApoE ε4 interfering species. Thus, it provides a viable tool for electrochemical determination of Aβ 1–42 . Spiked human and mice plasmas were used for the successful validation of the sensing platform in bio-fluidic samples. The results obtained from mice plasma analysis concurred with the immunohistochemistry (IHC) and magnetic resonance imaging (MRI) data obtained from brain analysis.Graphical abstract   Schematic representation of the electrochemical system proposed for Aβ 1–42  determination: (a) modification of graphene screen-printed electrode (SPE) with monolayer graphene oxide (GO) followed by its electrochemical reduction generating graphene/reduced graphene oxide (rGO) dual-layer (b), modification of dual-layer with linker (c), Aβ 1–42  antibody (H31L21) (d), bovine serum albumin (BSA) (e) and Aβ 1–42  peptide (f).Electronic supplementary material  The online version of this article (10.1007/s00604-020-04267-x) contains supplementary material, which is available to authorized users.

A label-free biosensor based on graphene and reduced graphene oxide dual-layer for electrochemical determination of beta-amyloid biomarkers