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

Abstract

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).

Keywords: Alzheimer’s disease; Aβ1–42 detection; Electrochemical biosensors; Graphene; Screen-printed electrodes.

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).

Fig. 1

Schematic representation of the electrochemical system for detection of Aβ_1–42: a graphene/rGO SPE-modified with linker (b), antibody (c), BSA (d) and Aβ_1–42 peptide (e)

Fig. 2

Raman spectra of graphene (blue) and graphene/rGO (black) dual-layer SPE

Fig. 3

Cyclic voltammograms for the modification of SPE with a graphene/rGO (a), graphene (b) and graphene/GO (c); b graphene/rGO (a), antibody (b), BSA (c) and linker (d), and the CV was taken in 1 M PBS containing 10 mM [Fe(CN)₆]³⁻ and 1 M KCl solution at a scan rate of 50 mV s⁻¹

Fig. 4

Scan rate studies of modified SPE a voltammograms under varying scan rates a-i (10, 25, 50, 75, 100, 125, 150, 175 and 200 mV s⁻¹); b anodic (Ipa) and cathodic (Ipc) peak currents versus the square root of corresponding scan rate

Fig. 5

Analytical performance of the biosensor a DPV curves obtained for the biosensor for detection of different concentration of Aβ_1–42 from a-h (0.2, 2, 11, 50, 220, 2200, 16,600 and 55,000 pM); b Calibration plot representing normalized current (I_C/I_blank) of DPV data as a function of Aβ_1–42 concentration on a logarithmic scale (n = 3)

Fig. 6

Specificity of the biosensor for the detection of 50 pM of Aβ_1–42 with 500 nM of interfering agents: Aβ_1–40 and ApoE ε4

Fig. 7

DPV responses from spiked concentration of Aβ_1–42 (50 (a), 220 (b), 2200 (c) and 16,600 (d) pM) in human plasma (a); calibration plot of normalized current (I_C/I_blank) versus log of Aβ_1–42 concentration (b); DPV responses for detection of WT (b) and Tg (c) mice compared with blank response (a); an age-based study with the two groups (9 and 12 months) of Tg animals (d) (n = 3)

Fig. 8

IHC data for the progression of AD pathology: An increase of human-specific Aβ_(1–42) (red) aggregation in cortex and hippocampal area; especially in stratum radiatum (SR), stratum lacunosum-moleculare (SLM) and outer portion of the molecular layer of dentate gyrus (OML) and the hilus of the dentate gyrus (DG), from 9 to12-months-old Tg compared with E littermates. Cornu ammonis (CA1, CA2, CA3 and hilus (CA4)) are subfield of the hippocampus; nuclei staining is in blue