A sensitive and selective electrochemical biosensor for the determination of beta-amyloid oligomer by inhibiting the peptide-triggered in situ assembly of silver nanoparticles

Abstract

Soluble beta-amyloid (Aβ) oligomer is believed to be the most important toxic species in the brain of Alzheimer's disease (AD) patients. Thus, it is critical to develop a simple method for the selective detection of Aβ oligomer with low cost and high sensitivity. In this paper, we report an electrochemical method for the detection of Aβ oligomer with a peptide as the bioreceptor and silver nanoparticle (AgNP) aggregates as the redox reporters. This strategy is based on the conversion of AgNP-based colorimetric assay into electrochemical analysis. Specifically, the peptide immobilized on the electrode surface and presented in solution triggered together the in situ formation of AgNP aggregates, which produced a well-defined electrochemical signal. However, the specific binding of Aβ oligomer to the immobilized peptide prevented the in situ assembly of AgNPs. As a result, a poor electrochemical signal was observed. The detection limit of the method was found to be 6 pM. Furthermore, the amenability of this method for the analysis of Aβ oligomer in serum and artificial cerebrospinal fluid (aCSF) samples was demonstrated.

Keywords: Alzheimer’s disease; beta-amyloid oligomer; electrochemical biosensors; peptide; silver nanoparticles.

Figure 1

Characterization of AgNPs in the presence PrP_95–110 or Aβ oligomer/PrP_95–110. Notes: (A) UV–Vis absorption spectra of AgNPs in the absence and presence of PrP_95–110 or Aβ oligomer/PrP_95–110. TEM images of AgNPs in the presence of PrP_95–110 (B) or Aβ oligomer/PrP_95–110 (C). The concentrations of AgNPs, PrP_95–110 and Aβ sample (equivalent monomer) were 2.4 nM, 0.1 μM and 2 μM, respectively. Abbreviations: UV, ultraviolet; Vis, visible; AgNP, silver nanoparticle; PrP, prion protein; Aβ, beta-amyloid; TEM, transmission electron microscope.

Figure 2

The LSV responses of the PrP_95–110-functionalized electrodes after incubation with AgNPs/PrP_95–110 (black curve), PrP_95–110 (red curve), AgNPs (blue curve), Aβ oligomer and AgNPs/PrP_95–110 (green curve) and the mixture of Aβ oligomer/PrP_95–110/AgNPs (magenta curve). Notes: The arrow indicates the scan direction. The concentrations of AgNPs, PrP_95–110 and Aβ sample were 2.4 nM, 0.1 μM and 2 μM, respectively. Abbreviations: LSV, linear sweep voltammetry; PrP, prion protein; AgNP, silver nanoparticle; Aβ, beta-amyloid.

Figure 3

Dependence of the current on the concentration ratio of PrP_95–110 to AgNPs (A) and the AgNPs concentration (B). Notes: In (A), the AgNPs concentration was kept at 2.4 nM and the concentration of PrP_95–110 was increased from 0.05 to 0.5 μM (0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μM). In (B), the concentration ratio of PrP_95–110 to AgNPs was kept at 83:1 and the AgNPs concentration was increased from 0.15 to 4.8 nM (0.15, 0.3, 0.45, 0.9, 1.2, 2.4 and 4.8 nM). I_pa, oxidation current. Abbreviations: PrP, prion protein; AgNP, silver nanoparticle; Aβ, beta-amyloid.

Figure 4

Influence of Aβ incubation time on the formation of Aβ oligomer and the current. Notes: (A) Dependence of the current on the incubation time for the preparation of Aβ oligomer. The final concentrations of AgNPs, PrP_95–110 and Aβ sample were kept at 1.2 nM, 100 nM and 1 μM, respectively. (B) AFM images of the mica substrate after incubation with Aβ samples pre-incubated for 24 and 48 h. I_pa, oxidation current. Abbreviations: Aβ, beta-amyloid; AgNP, silver nanoparticle; PrP, prion protein; AFM, atomic force microscopy.

Figure 5

Sensitivity and selectivity. Notes: (A) Dependence of the current on the concentration of Aβ sample (0.01, 0.2, 5, 50, 200, 500 and 2,000 nM). The inset shows the linear dependence of the current change on the concentration of the Aβ sample. (B) Selectivity of the sensing protocol (bar 1, Aβ monomer; bar 2, Aβ fibril; bar 3, Aβ oligomer; bar 4, BSA; bar 5, IgG and bar 6, thrombin). The concentration of the Aβ sample was 200 nM and that of BSA, IgG and thrombin was 1 μM. I_pa, oxidation current. Abbreviations: Aβ, beta-amyloid; BSA, bovine serum albumin; IgG, immunoglobin G.

Scheme 1

Schematic illustration of the previous electrochemical strategies for the detection of Aβ oligomer with PrP_95–110 as the receptor and AgNP aggregates as the redox reporters. Abbreviations: Aβ, beta-amyloid; PrP, prion protein; AgNP, silver nanoparticle; Ad, adamantine; β-CD, β-cyclodextrin.

Scheme 2

Schematic illustration of the present electrochemical strategies for the detection of Aβ oligomer with PrP_95–110 as the receptor and AgNP aggregates as the redox reporters. Abbreviations: Aβ, beta-amyloid; PrP, prion protein; AgNP, silver nanoparticle.