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

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 (aβagg) were used. Electrochemical impedance spectroscopy was used to probe the changes in charge transfer resistance (Rct) of the immunosensor after the specific binding of biosensor with aβagg. The subsequent incubation of the aβagg complex with a specific concentration of EPPS at different time intervals divulged AD progression. The decline in the Rct of the immunosensor started at 10 min of EPPS incubation and continued to decrease gradually from 20 min, indicating that the accumulation of aβagg on the surface of the anti-aβ/SAM/ICE sensor has been extricated. Here, the kinetic disaggregation rate k value of aβagg was found to be 0.038. This innovative study using electrochemical measurement to investigate the mechanism of aβagg disaggregation by EPPS could provide a new perspective in monitoring the disaggregation periods of aβagg from oligomeric to monomeric form, and then support for the prediction and handling AD symptoms at different stages after treatment by a drug, EPPS.


Introduction
The deposition of the amyloid-β (aβ) protein in the cortex of the brain is reported as a leading cause of Alzheimer's disease (AD), the most common form of dementia [1]. According to the amyloid cascade hypothesis, aβ peptides tend to aggregate and coalesce into toxic insoluble oligomers and fibrils in plaques, which may cause decline in synaptic function and nerve cell damage [2,3].
Aggregation of aβ gradually increases throughout the brain, which leads to neurodegeneration, starting from mild to moderate symptoms in earlier Alzheimer's stages, and finally dementia [3,4]. Many efforts have been made to control and prevent Alzheimer's progression by inhibiting and reducing the aggregation of aβ. For instance, laser-induced
De-ionized (DI) water is gained from Milli-Q system. Phosphate buffer saline (PBS, pH 7.4) is received from Tech-Innovation.

Development of the Immunosensor for Monitoring the Impact of EPPS on the Disaggregation of aβ agg
On a glass transparency substrate (13.5 × 16.0 mm), an interdigitated chain-shaped electrode (ICE) is made. Ti and Au have been deposited in an electron beam-evaporator with electrodes 25 nm and 50 nm of thickness. The lift-off procedure then formed the coupled electrode finger consisting of 5 µm distance and width for working and reference electrodes. The ICE was then cleaned with 100% ethanol, DI water, and dried in nitrogen gas flow to prepare for the development of the immunosensor. Schematic of the fabrication and microscope image of ICE are displayed in Figure 1.  The development of the immunosensor for diagnosis of aβagg is displayed in Figure  2. A self-assembled monolayer (SAM) has been mounted on the purified ICE gold surface in 100 mM MHA for 24 hours at room temperature. A solution of 75 mM EDC and 5 mM NHS was then incubated with the SAM-modified electrode to activate the carboxyl group for antibody binding. Then, ten anti-aβ antibody microliters (100 μg·mL −1 ) in PBS (pH ~ 7.4) are dripped off into a modified electrode and incubated for 1 hour in a moist compartment at 4 °C. Therefore, the EDC/NHS-activated SAM molecules on the modified electrodes were attached to the anti-aβ antibody through the amino group. After that, nonspecific adsorption was blocked by incubating with BSA (0.5% in 1 × PBS, pH 7.4) for 20 min at 4 °C to develop the anti-aβ/SAM/ICE biosensor. The developed anti-aβ/SAM/ICE biosensor was then incubated with aβagg (in PBS (pH ~ 7.4)) for 20 min at room temperature for the recognition of aβagg. The development of the immunosensor for diagnosis of aβ agg is displayed in Figure 2. A self-assembled monolayer (SAM) has been mounted on the purified ICE gold surface in 100 mM MHA for 24 hours at room temperature. A solution of 75 mM EDC and 5 mM NHS was then incubated with the SAM-modified electrode to activate the carboxyl group for antibody binding. Then, ten anti-aβ antibody microliters (100 µg·mL −1 ) in PBS (pH~7.4) are dripped off into a modified electrode and incubated for 1 hour in a moist compartment at 4 • C. Therefore, the EDC/NHS-activated SAM molecules on the modified electrodes were attached to the anti-aβ antibody through the amino group. After that, nonspecific adsorption was blocked by incubating with BSA (0.5% in 1 × PBS, pH 7.4) for 20 min at 4 • C to develop the anti-aβ/SAM/ICE biosensor. The developed anti-aβ/SAM/ICE biosensor was then incubated with aβ agg (in PBS (pH~7.4)) for 20 min at room temperature for the recognition of aβ agg .
To monitor the effect of EPPS on the kinetics disaggregation of aβ agg , the antiaβ/SAM/ICE biosensor was incubated with aβ agg (2.5 × 10 −3 mg·mL −1 ) for 20 min to enable a specific binding between the antibody and antigen, called the aβ agg complex. Subsequently, the aβ agg complex was incubated with 20 mM·L −1 of EPPS for various time periods to study the disaggregation process of aβ agg by electrochemical impedance measurement. To monitor the effect of EPPS on the kinetics disaggregation of aβagg, the antiaβ/SAM/ICE biosensor was incubated with aβagg (2.5 × 10 −3 mg·mL −1 ) for 20 min to enable a specific binding between the antibody and antigen, called the aβagg complex. Subsequently, the aβagg complex was incubated with 20 mM·L −1 of EPPS for various time periods to study the disaggregation process of aβagg by electrochemical impedance measurement.

EIS Measurements and Functional Group Characterization
EIS was performed using EC-Lab (SP-200, Bio-Logic, France) in 5 mM [Fe(CN)6] 3‾/4‾ in PBS (pH 7.4) at each step of the anti-aβ/SAM/ICE biosensor development and various time intervals of the disaggregation process of aβagg. The EIS data was obtained by putting on a 10 mV of sinus amplitude which satisfy the linear properties of electrical response of the object between the working and reference electrode from 100 mHz to 1 MHz of range frequency. Z-Fit software (EC-Lab, Bio-Logic, France) was used to fit the obtained EIS data through by the Randle's equivalent circuit.
Fourier-transform infrared (FT-IR) spectra was characterized by using Jasco-4600 FTIR (USA) spectrometer to confirm the functional groups for the fabrication steps of the anti-aβ/SAM/ICE biosensor.

Investigating the Development of Impedimetric Immunosensor
As shown in Figure 3a, EIS measurements were carried out to determine the Nyquistplots with the imaginary impedance component (-Im(Z)) plotted with the actual impedance component (Re(Z)) to confirm the successful modification of substantial ingredients to the electrode surface at different stages of the development the anti-aβ/SAM/ICE immunosensor.
The equivalent circuit model of Randle insert into Figure 3a was used to fit the Nyquist plot data, showing three EIS parameters, including the CPE (constant electrodesolution interface element), Rct (interfacial charge-transfer resistance that corresponds to the semicircular diameter in the Nyquist plot), and Rs (solution resistance).  Fourier-transform infrared (FT-IR) spectra was characterized by using Jasco-4600 FTIR (USA) spectrometer to confirm the functional groups for the fabrication steps of the anti-aβ/SAM/ICE biosensor.

Investigating the Development of Impedimetric Immunosensor
As shown in Figure 3a, EIS measurements were carried out to determine the Nyquistplots with the imaginary impedance component (-Im(Z)) plotted with the actual impedance component (Re(Z)) to confirm the successful modification of substantial ingredients to the electrode surface at different stages of the development the anti-aβ/SAM/ICE immunosensor. The obtained fitting value of Rct from Figure 3a was displayed in Figure 3b and Table  1. After SAM deposition that can be attributed to the −COO‾ group and the carbon chain SAM which prevents the transfer of the negative charged [Fe(CN)6] 3‾/4‾ redox couple to the electrode surface, the value of Rct has increased dramatically. Then, Rct decreased after the EDC-NHS layer was deposited on the SAM-modified electrode, suggesting that the −COO‾ groups of SAM were activated with the EDC-NHS carbodiimide coupler, the formation of succinimide ester on the modified electrode surface of SAM allows fast electron transfer between the electrode and the interface [19]. The two continuous immobilization steps of anti-aβ and BSA showed an increase in Rct due to the binding of a specific material on the sensor surface which enhanced its surface resistance [20], demonstrating that the surface of the biosensor was covered by specific materials.    The equivalent circuit model of Randle insert into Figure 3a was used to fit the Nyquist plot data, showing three EIS parameters, including the C (capacitance for the electrodesolution interface element), R ct (interfacial charge-transfer resistance that corresponds to the semicircular diameter in the Nyquist plot), and R s (solution resistance).
The obtained fitting value of R ct from Figure 3a was displayed in Figure 3b and Table 1. After SAM deposition that can be attributed to the −COO − group and the carbon chain SAM which prevents the transfer of the negative charged [Fe(CN) 6 ] 3−/4− redox couple to the electrode surface, the value of R ct has increased dramatically. Then, R ct decreased after the EDC-NHS layer was deposited on the SAM-modified electrode, suggesting that the −COO − groups of SAM were activated with the EDC-NHS carbodiimide coupler, the formation of succinimide ester on the modified electrode surface of SAM allows fast electron transfer between the electrode and the interface [19]. The two continuous immobilization steps of anti-aβ and BSA showed an increase in R ct due to the binding of a specific material on the sensor surface which enhanced its surface resistance [20], demonstrating that the surface of the biosensor was covered by specific materials. Fourier-transform infrared (FT-IR) spectra was also used to confirm the fabrication of the anti-aβ/SAM/ICE biosensor, and the results were displayed in Figure 4. The FT-IR result of the SAM-modified ICE showed the typical peaks near 1601 cm −1 of carbonyl from the carboxylic acid moiety on the alkanethiols of SAM [21], indicating the immobilization of SAM on the ICE. FT-IR result of the immobilized anti-aβ on the EDC-NHS coupler activated SAM-modified ICE (blue curve in Figure 4) showed the typical peaks of protein at near 3374 cm −1 of N-H stretching of amide II [22], 2925 and 2849 cm −1 of C-H stretching of -CH 2 of protein [23,24], 920 cm −1 of C-O stretching [25], and 602 cm −1 of N-H deformation [22], which demonstrated the successful development of the anti-aβ/SAM/ICE biosensor.

Monitoring the Effect of EPPS on aβ Disaggregation
EPPS was used to target the disaggregation of aβagg into its monomeric form. To confirm whether EPPS had an effect on the disaggregation of aβagg, EIS was used to explore the change in Rct of the specific anti-aβ/SAM/ICE biosensor for recognizing aβ during incubation with EPPS at different time intervals.

Monitoring the Effect of EPPS on aβ Disaggregation
EPPS was used to target the disaggregation of aβ agg into its monomeric form. To confirm whether EPPS had an effect on the disaggregation of aβ agg , EIS was used to explore the change in R ct of the specific anti-aβ/SAM/ICE biosensor for recognizing aβ during incubation with EPPS at different time intervals.
Firstly, the impact of EPPS on the anti-aβ antibody was studied. EIS measurements of the anti-aβ/SAM/ICE biosensor after incubation with 20 mM·L −1 EPPS at different time intervals are displayed in Figure 5. The Nyquist plots in Figure 5a did not change significantly after treatment with EPPS. Using Randle's equivalent circuit to fit the EIS data of Nyquist plots in Figure 5a, the R ct value was generated that serve to normalize the response of the anti-aβ/SAM/ICE biosensor to the EPPS treatment at 10, 20, 30, 40, 50 min in Figure 5b, where R 0 represents the R ct value of the biosensor with the immobilization of anti-aβ antibody, and R a represents the R ct value of the anti-aβ/SAM/ICE biosensor within the treatment of EPPS. The normalized value (R a − R 0 )/R 0 of the anti-aβ/SAM/ICE biosensor in Figure 5b did not change significantly after treatment with EPPS at various time periods, indicating that EPPS did not affect the anti-aβ antibody. The fitting EIS data were shown in Table 2.   Figure 5a to the equivalent circuit model of Randle.  [26] was used to confirm the aggregation state of aβ in this manuscript. As seen in Figure 6, the gel electrophoresis result of aβagg showed the molecular-weight ranging of 11 kDa for oligomer form, which corresponds to a smear band containing monomers and low-molecular-weight oligomers ranging between 4 and 18 kDa in size of aβ oligomers [27], demonstrating the oligomeric form of aβagg in this research.  Table 2. EIS parameters of the anti-aβ/SAM/ICE biosensor after treatment with EPPS at different time intervals, extrapolated by fitting the measured spectra in Figure 5a to the equivalent circuit model of Randle. Gel electrophoresis method [26] was used to confirm the aggregation state of aβ in this manuscript. As seen in Figure 6, the gel electrophoresis result of aβ agg showed the molecular-weight ranging of 11 kDa for oligomer form, which corresponds to a smear band containing monomers and low-molecular-weight oligomers ranging between 4 and 18 kDa in size of aβ oligomers [27], demonstrating the oligomeric form of aβ agg in this research.  [26] was used to confirm the aggregation state of aβ in this manuscript. As seen in Figure 6, the gel electrophoresis result of aβagg showed the molecular-weight ranging of 11 kDa for oligomer form, which corresponds to a smear band containing monomers and low-molecular-weight oligomers ranging between 4 and 18 kDa in size of aβ oligomers [27], demonstrating the oligomeric form of aβagg in this research. The process of disaggregation of aβagg by EPPS at different time intervals is described in Section 2.2. Initially, the anti-aβ/SAM/ICE biosensor was incubated with aβagg (2.5 × 10 −3 mg·mL −1 ) for 20 min in the absence of EPPS and measured via EIS to examine the aβ recognition of the biosensor. EIS measurements showed an increase in the diameter and height of the semicircle in the Nyquist plot, corresponding to the enhanced Rct after the anti- The process of disaggregation of aβ agg by EPPS at different time intervals is described in Section 2.2. Initially, the anti-aβ/SAM/ICE biosensor was incubated with aβ agg (2.5 × 10 −3 mg·mL −1 ) for 20 min in the absence of EPPS and measured via EIS to examine the aβ recognition of the biosensor. EIS measurements showed an increase in the diameter and height of the semicircle in the Nyquist plot, corresponding to the enhanced R ct after the anti-aβ/SAM/ICE biosensor was incubated with aβ agg , which is marked by 0 min in Figure 7a. This enhancement of R ct is ascribed to the hindrance of electron transfer between the [Fe(CN) 6 ] 3−/4− redox couple and the electrode surface due to successful specific binding between anti-aβ antibody and aβ agg , called aβ agg complex on the surface of the sensor. The biosensor with the aβ agg complex was subsequently incubated for different time periods (10,20,30,40, and 50 min) with 20 mM·L −1 of EPPS and was used for EIS measurements. As shown in Figure 7a, after 10 min of incubation in EPPS, the R ct of the biosensor with the aβ agg complex in the Nyquist plot showed an increase in the height and diameter of the semicircle, and the Nyquist plot remained the same for the next 20 min of incubation with EPPS, which indicated that the [Fe(CN) 6 ] 3−/4− ion transport towards the electrode surface was blocked due to excessive aβ on the electrode surface. These results implied that the disaggregation of aβ agg started after 10 min of incubation with EPPS and was continuously maintained after 20 min of incubation. This disaggregation process converted the aβ agg into small aβ monomers, and still retained a certain amount of aβ agg , creating an abundant compound of aβ monomers and aβ agg on the biosensor surface, as delineated in Figure 8a, which hindered the transportation of [Fe(CN) 6 ] 3−/4− redox couple to the electrode surface, leading to an increase in R ct . Figure 7a showed the decline in R ct after treatment with EPPS at 30 min, which manifested the disaggregation of the remaining aβ agg into several monomeric aβ molecules on the sensor surface, suggesting that the movement of ion [Fe(CN) 6 ] 3−/4− toward the electrode surface was facilitated; the impeded ion [Fe(CN) 6 ] 3−/4− movement was caused by the presence of a dense aβ agg that was reduced by converting the aβ agg molecules into aβ monomers, leading to the expansion of spacing between the specific aβ molecules on the electrode surface, resulting in facilitating transport of redox couple [Fe(CN) 6 ] 3−/4− . This decline of R ct continued to occur at 40 and 50 min during EPPS treatment, which indicated the continuous disaggregation of aβ agg . A graphical description of the disaggregation process of aβ agg by EPPS at various time intervals is shown in Figure 8a. The response of aβ agg to EPPS treatment at time periods was evaluated by normalizing the R ct value of Figure 7a and plotted against time, as (R b − R i )/R i in Figure 7b, where R i represents the R ct of the biosensor with the immobilization of aβ agg , and R b represents the R ct of the biosensor with the immobilization of aβ agg within the EPPS treatment. The normalized value (R b − R i )/R i in Figure 7b increased after 10 min of EPPS treatment, remained stable after 20 min, and gradually decreased over the 30, 40, and 50 min points, which indicated the disaggregation process of aβ agg at different time intervals starting at 10 min and confirmed the effect of EPPS on aβ agg disag- gregation, corresponding to the results and explanation in Figure 7a. A detailed graphical description of the disaggregation process of aβ agg by EPPS at various time intervals is provided in Figure 8a. The fitting EIS data were shown in Table 3.

Electrode R s (Ω) C (F) R ct (Ω)
shown in Figure 8a. The response of aβagg to EPPS treatment at time periods was evaluated by normalizing the Rct value of Figure 7a and plotted against time, as (Rb-Ri)/Ri in Figure  7b, where Ri represents the Rct of the biosensor with the immobilization of aβagg, and Rb represents the Rct of the biosensor with the immobilization of aβagg within the EPPS treatment. The normalized value (Rb-Ri)/Ri in Figure 7b increased after 10 min of EPPS treatment, remained stable after 20 min, and gradually decreased over the 30, 40, and 50 min points, which indicated the disaggregation process of aβagg at different time intervals starting at 10 min and confirmed the effect of EPPS on aβagg disaggregation, corresponding to the results and explanation in Figure 7a. A detailed graphical description of the disaggregation process of aβagg by EPPS at various time intervals is provided in Figure 8a. The fitting EIS data were shown in Table 3.   Table 3. EIS parameters of the anti-aβ/SAM/ICE biosensor after incubating with aβagg, and then conduct to treatment with EPPS at different time intervals, extrapolated by fitting the measured spectra in Figure 7a to the equivalent circuit model of Randle.  The effect of EPPS on the aβ 1-42 peptide (aβpep), a long peptide of 1-42 amino acids, was also studied for comparison with the obtained results of aβagg, which are mentioned above. The experiment of aβpep treatment in EPPS was carried out through similar processes as described in Section 2 with aβagg replaced by aβpep. Nyquist plot of EIS result in Figure 9a show an increase in Rct of the biosensor anti-aβ/SAM/ICE after 20 min of incubation with aβpep. This increment of Rct is expected due to the repulsion between the negative charge of immobilized aβpep antigen on the anti-aβ/SAM/ICE biosensor with redox couple, that could prevent the redox couple from reaching the SAM-modified electrode, confirming the successful binding between anti-aβ and aβpep [28]. The Rct of the biosensor with the immobilized aβpep remained the same after 10 min of treatment in EPPS, indicating that aβpep was not affected by EPPS for the initial 10 min treatment. Rct significantly decreased after 20 and 30 min of subsequent EPPS treatment, indicating the facilitating electron transfer at the electrode interface. This decrease of Rct may be attributed to the fragmentation of aβpep by EPPS, leading to the formation of ion-complexed amyloid beta with [Fe(CN)6] 3‾/4‾ [29,30], which results in the decreasing of Rct and enhancing of electrochemical property of the sensor at 20 min treatment, and at more treatment time of 30 min [16]. The normalized result (Rb-Ri)/Ri is displayed in Figure 9b, where Ri represents the Rct of the biosensor with the immobilization of aβpep, and Rb represents the Rct of the biosensor with the immobilization of aβpep within the EPPS treatment, which corresponds with the data of Nyquist plots in Figure 9a. The (Rb-Ri)/Ri was stable after 10 min, and rapidly decreased over the 20 and 30 min points following EPPS treatment. Figure 8b shows the effect of EPPS treatment on aβpep, and EIS results are shown in Figure 9. The fitting EIS data were shown in Table 4.  Table 3. EIS parameters of the anti-aβ/SAM/ICE biosensor after incubating with aβ agg , and then conduct to treatment with EPPS at different time intervals, extrapolated by fitting the measured spectra in Figure 7a to the equivalent circuit model of Randle. The effect of EPPS on the aβ 1-42 peptide (aβ pep ), a long peptide of 1-42 amino acids, was also studied for comparison with the obtained results of aβ agg , which are mentioned above. The experiment of aβ pep treatment in EPPS was carried out through similar processes as described in Section 2 with aβ agg replaced by aβ pep . Nyquist plot of EIS result in Figure 9a show an increase in R ct of the biosensor anti-aβ/SAM/ICE after 20 min of incubation with aβ pep . This increment of R ct is expected due to the repulsion between the negative charge of immobilized aβ pep antigen on the anti-aβ/SAM/ICE biosensor with redox couple, that could prevent the redox couple from reaching the SAMmodified electrode, confirming the successful binding between anti-aβ and aβ pep [28]. The R ct of the biosensor with the immobilized aβ pep remained the same after 10 min of treatment in EPPS, indicating that aβ pep was not affected by EPPS for the initial 10 min treatment. R ct significantly decreased after 20 and 30 min of subsequent EPPS treatment, indicating the facilitating electron transfer at the electrode interface. This decrease of R ct may be attributed to the fragmentation of aβ pep by EPPS, leading to the formation of ion-complexed amyloid beta with [Fe(CN) 6 ] 3−/4− [29,30], which results in the decreasing of R ct and enhancing of electrochemical property of the sensor at 20 min treatment, and at more treatment time of 30 min [16]. The normalized result (R b − R i )/R i is displayed in Figure 9b, where R i represents the R ct of the biosensor with the immobilization of aβ pep , and R b represents the R ct of the biosensor with the immobilization of aβ pep within the EPPS treatment, which corresponds with the data of Nyquist plots in Figure 9a. The (R b − R i )/R i was stable after 10 min, and rapidly decreased over the 20 and 30 min points following EPPS treatment. Figure 8b shows the effect of EPPS treatment on aβ pep , and EIS results are shown in Figure 9. The fitting EIS data were shown in Table 4.  Table 4. EIS parameters of the anti-aβ/SAM/ICE biosensor after incubating with aβpep, and then conduct to treatment with EPPS at different time intervals, extrapolated by fitting the measured spectra in Figure 9a to the equivalent circuit model of Randle. 129,281 By examining the EIS results of aβagg and aβpep in the treatment with EPPS in this study, it is observed that EPPS affects the disaggregation of aβagg gradually and leads to the establishment of a disaggregation mechanism, as shown in Figure 8, while EPPS causes rapid disruption of aβpep.

Kinetic Disaggregation Rate k
The kinetic disaggregation rate k of protein is an important parameter in evaluating the functioning of neurodegenerative diseases [31]. However, there have not been report on the k value for aβagg at present. To estimate the disaggregation rate k value for aβagg, the normalized data for the treatments of aβagg and aβpep in EPPS were plotted and fitted versus time, as shown in Figure 10. Data for the disaggregation experiment aβagg represent the best fit of the data to a single-exponential function y=0.149−0.048exp(0.038x), whereas data for the treatment aβpep in EPPS express a linear fit function y=0.271−0.027x. According to the studies of kinetic disaggregation of proteins, the fitting data for protein disaggrega-  Table 4. EIS parameters of the anti-aβ/SAM/ICE biosensor after incubating with aβ pep , and then conduct to treatment with EPPS at different time intervals, extrapolated by fitting the measured spectra in Figure 9a to the equivalent circuit model of Randle. By examining the EIS results of aβ agg and aβ pep in the treatment with EPPS in this study, it is observed that EPPS affects the disaggregation of aβ agg gradually and leads to the establishment of a disaggregation mechanism, as shown in Figure 8, while EPPS causes rapid disruption of aβ pep .

Kinetic Disaggregation Rate k
The kinetic disaggregation rate k of protein is an important parameter in evaluating the functioning of neurodegenerative diseases [31]. However, there have not been report on the k value for aβ agg at present. To estimate the disaggregation rate k value for aβ agg , the normalized data for the treatments of aβ agg and aβ pep in EPPS were plotted and fitted versus time, as shown in Figure 10. Data for the disaggregation experiment aβ agg represent the best fit of the data to a single-exponential function y = 0.149 − 0.048exp(0.038x), whereas data for the treatment aβ pep in EPPS express a linear fit function y = 0.271 − 0.027x. According to the studies of kinetic disaggregation of proteins, the fitting data for protein disaggregation has been expressed by an exponential function F = y 0 + Aexp(−kt) [31,32], where k represents the disaggregation rate. Consequently, the disaggregation rate k of aβ agg was determined to be 0.038 by our method, providing a reference parameter for the disaggregation of aβ agg for future studies.

Conclusions
A new concept of measuring aβagg disaggregation following treatment with EPPS via EIS signaling was introduced in this report. An electrochemical impedimetric immunosensor was developed to monitor EIS signals at the disaggregation stages of aβagg during treatment with EPPS at different time intervals. The obtained EIS results confirmed that the disaggregation of aβagg by treatment in EPPS occurred after 10 min, and this disaggregation process continued gradually over the next 50 min. A single exponential function for aβagg disaggregation was also found in this report, with a kinetic disaggregation rate (k) value of 0.038. The data indicated the successful application of EIS measurement in monitoring and establishing the mechanism for the impact of EPPS on the disaggregation of aβagg, which may lead to new insights in the diagnosis and treatment of signs of AD at various phases.

Conclusions
A new concept of measuring aβ agg disaggregation following treatment with EPPS via EIS signaling was introduced in this report. An electrochemical impedimetric immunosensor was developed to monitor EIS signals at the disaggregation stages of aβ agg during treatment with EPPS at different time intervals. The obtained EIS results confirmed that the disaggregation of aβ agg by treatment in EPPS occurred after 10 min, and this disaggregation process continued gradually over the next 50 min. A single exponential function for aβ agg disaggregation was also found in this report, with a kinetic disaggregation rate (k) value of 0.038. The data indicated the successful application of EIS measurement in monitoring and establishing the mechanism for the impact of EPPS on the disaggregation of aβ agg , which may lead to new insights in the diagnosis and treatment of signs of AD at various phases.