Deciphering the Disaggregation Mechanism of Amyloid Beta Aggregate by 4-(2-Hydroxyethyl)-1-Piperazinepropanesulfonic Acid Using Electrochemical Impedance Spectroscopy

Abstract

Aggregation of amyloid-β (aβ) peptides into toxic oligomers, fibrils, and plaques is central in the molecular pathogenesis of Alzheimer's disease (AD) and is the primary focus of AD diagnostics. Disaggregation or elimination of toxic aβ aggregates in patients is important for delaying the progression of neurodegenerative disorders in AD. Recently, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) was introduced as a chemical agent that binds with toxic aβ aggregates and transforms them into monomers to reduce the negative effects of aβ aggregates in the brain. However, the mechanism of aβ disaggregation by EPPS has not yet been completely clarified. In this study, an electrochemical impedimetric immunosensor for aβ diagnostics was developed by immobilizing a specific anti-amyloid-β (aβ) antibody onto a self-assembled monolayer functionalized with a new interdigitated chain-shaped electrode (anti-aβ/SAM/ICE). To investigate the ability of EPPS in recognizing AD by extricating aβ aggregation, commercially available aβ aggregates (aβ_agg) were used. Electrochemical impedance spectroscopy was used to probe the changes in charge transfer resistance (R_ct) of the immunosensor after the specific binding of biosensor with aβ_agg. The subsequent incubation of the aβ_agg complex with a specific concentration of EPPS at different time intervals divulged AD progression. The decline in the R_ct of the immunosensor started at 10 min of EPPS incubation and continued to decrease gradually from 20 min, indicating that the accumulation of aβ_agg on the surface of the anti-aβ/SAM/ICE sensor has been extricated. Here, the kinetic disaggregation rate k value of aβ_agg was found to be 0.038. This innovative study using electrochemical measurement to investigate the mechanism of aβ_agg disaggregation by EPPS could provide a new perspective in monitoring the disaggregation periods of aβ_agg from oligomeric to monomeric form, and then support for the prediction and handling AD symptoms at different stages after treatment by a drug, EPPS.

Keywords: 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; Alzheimer’s disease; amyloid beta aggregate; electrochemical impedance spectroscopy; impedimetric immunosensor; kinetic disaggregation of protein.

Figure 1

(a) Fabrication of ICE, (b) Photograph of ICE, and (c) Scanning electron microscope image of chain-shaped gold finger of ICE.

Figure 2

(a–e) Development procedure the immunosensor anti-aβ/SAM/ICE for the recognition aβ_agg, (f) impedimetric measurement mechanism in the recognition aβ_agg by the developed biosensor anti-aβ/SAM/ICE, and (g) impedimetric results represent by Nyquist plot at each step of the development anti-aβ/SAM/ICE biosensor in the recognition aβ_agg.

Figure 3

EIS results at each step of the development of biosensor anti-aβ/SAM/ICE display in (a) Nyquist plots and (b) charge-transfer resistance (R_ct).

Figure 4

FT-IR results for the fabrication of the anti-aβ/SAM/ICE biosensor.

Figure 5

EIS results of the biosensor anti-aβ/SAM/ICE with the treatment in EPPS at different time intervals display in (a) Nyquist plots and (b) Normalization of R_ct. (n = 3; three data points shown).

Figure 6

Gel electrophoresis results of aβ_pep and aβ_agg.

Figure 7

EIS results monitoring the disaggregation aβ_agg with the treatment in EPPS at different time intervals display in (a) Nyquist plots and (b) Normalization of R_ct. (n = 3; three data points shown).

Figure 8

(a) Disaggregation mechanism of aβ_agg by EPPS, and (b) the impact of EPPS on aβ_pep.

Figure 9

EIS results monitoring the impact of EPPS on the aβ_pep at different time intervals display in (a) Nyquist plots and (b) Normalization of R_ct. (n = 3; three data points shown).

Figure 10

Fitting of two sets of disaggregation data for aβ_agg and aβ_pep, respectively; symbols and bars represent the average and standard deviation of the data (n = 3).