Simultaneous Monitoring of Amyloid-β (Aβ) Oligomers and Fibrils for

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Simultaneous Monitoring of A# Oligomers and Fibrils for Effectively Evaluating the Dynamic Process of A# Aggregation Yanyan Yu, Tianxiao Yin, Qiwen Peng, Lingna Kong, Chenglin Li, Daoquan Tang, and Xiaoxing Yin ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01493 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Simultaneous Monitoring of Aβ Oligomers and Fibrils for Effectively Evaluating the Dynamic Process of Aβ Aggregation Yanyan Yu,1,2,‡ Tianxiao Yin,1,‡ Qiwen Peng,1 Lingna Kong,3 Chenglin Li,1 Daoquan Tang,1,2,* Xiaoxing Yin1,* 1Jiangsu

Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, P.R.China 2

Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, P.R.China 3 Department

of Chemistry, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P. R. China

KEYWORDS: Alzheimer’s disease; Aβ oligomers; Aβ fibrils; Simultaneous; Dynamic.

ABSTRACT: Herein, we showed proof of concept for a novel strategy that targeted the assessment of aggregation process of amyloid-β (Aβ) by simultaneously determining its oligomers (Aβo) and fibrils (Aβf) in one analytical system. By fabricating and combining two immunosensors for Aβo and Aβf, respectively, a two-channel electrochemical system was constructed. The ratio of Aβf versus Aβo was calculated and taken as a possible criteria for evaluating the aggregation extent. Thereby, the presence and transformation between oligomers and fibrils was accurately probed by incubating Aβ monomer for different time and then calculating the ratios of Aβf to Aβo. The applicability of this method was further validated by tracking the dynamic progress of Aβ aggregation in CSF and tissues of AD rats, which revealed that the ratio of Aβf to Aβo in rat brain gradually rose in the time course of AD, indicative of the severity of peptide aggregation during this process. Overall, this study represented the first example of a quantitative strategy for precisely evaluating the aggregation process that was related to pathological events in AD brain.

Alzheimer's disease (AD) is a common primary degenerative disease of the central nervous system. Its clinical manifestations mainly include progressive and near-memory disorders, analytical and judgment abilities, cognitive disorders and disorders.1 It has been estimated that the possibility of suffering from AD will increase significantly after the age of 70, and it may affect around 50% of persons over the age of 85,2 which prompt the urgent development of novel analytical strategies capable of targeting pathogenic factors directly linked to neurodegeneration. Although the explicit justification causing AD pathogenicity remains to be discovered, it has been widely acknowledged that extracellular plaques, mainly composed of amyloid-β peptide (Aβ) species, and intracellular neurofibrillary tangles made of hyperphosphorylated tau protein are the two most prominent histopathological hallmarks of AD.3,4 Multiple evidences have indicated that the accumulation of Aβ in the brain contributes to the loss of neurons and subsequent development of AD pathology.5,6 Aβ is a small peptide fragment of 39-43 amino acids with a molecular weight of 4-kDa, which is a transmembrane secretory product of amyloid precursor protein (APP).7 A considerable amount of knowledge on the molecular etiology of AD involves the aggregation of Aβ peptide-40/42-residue

fragments of Aβ produced from sequentially proteolytic cleavage of amyloid precursor protein (APP) by β- and γsecretases under certain conditions.8-10 According to the original amyloid-cascade hypothesis, Aβ monomers will gradually self-aggregate to form neurotoxic oligomer oligomers, and as the disease progresses, the oligomers will further aggregate to form insoluble fiber filaments, both of which are neurotoxic and cause death of brain cells.11,12 Elevated levels of Aβ oligomers have been found in the cerebrospinal fluids of AD patients.13,14 However, soluble, oligomeric forms of Aβ are the most toxic species, rather than more aggregated fibrils or protofibrils.15-17 The possible mechanism of Aβ oligomers – induced toxicity may be due to their incorporation into the lipid bilayer of neuronal cells, resulting from unrestricted influx of Ca2+ ions and excessive membrane depolarization.18-20 Fibrous aggregation of Aβ and amyloid precursor protein (APP) and other transmembrane receptors in the cell surface and the secretion pathway interaction, cross-linking, leading to inhibition of signaling pathways and abnormal activation, which start the nerve cell death program.21 Together, these studies suggest that a more accurate and quantitative evaluation on the dynamic formation of both oligomeric and fibrillar aggregates in the development of AD is urgently

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needed to explore the mechanism of action of Aβ on neurotoxicity in AD. However, due to the short lifetime, low concentration of Aβ oligomers and fibrils, and the lack of suitable methods to separate them from high molecular weight Aβ, the direct identification and simultaneous determination of Aβ oligomers and fibrils is still a great challenge but will have crucial clinical significance. Currently, polyacrylamide gel electrophoresis, size exclusion chromatography and fluorescence depolarization rate and other methods have been successfully used for the detection of early aggregation phase intermediates.22-25 However, these methods are still only based on the qualitative basis, which cannot reflect the dynamic change of contents of Aβ aggregates in the pathogenesis of AD. Alternatively, electrochemical biosensors have received more and more attentions because of the striking advantages of simplicity, selectivity, low instrumental cost, and capability in real-time, even in vivo detection.26-28 Immunoassay based on the antibody-antigen interaction is one of the most important electrochemical techniques in the quantitative detection of important species. Typically, analytes on the sensor are detected by using a specific antibody and labels in immunoassay, and the concentration of analytes is generally achieved by detecting the amount of labels.29 So far, protein recognition based on double-antibody sandwiches is a commonly used method in quantitative immunoassays.30,31 Similarly, in our present contribution, two immunosensors based on "sandwich strategy" were fabricated and accordingly, a two-channel electrochemical system was constructed to target the simultaneous determination of Aβ oligomers (Aβo) and its fibrils (Aβf) (Scheme 1). In this system, thionine (Th) was adopted as the signal amplification element, which was linked in a bioconjugate prepared by covalently binding a polyclonal antibody (A11 or OC) that can capture Aβo and Aβf via specific interactions with nanostructured lipid carrier (NLC). NLC has attracted increasing attentions because of their limited toxicity, good physiochemical stability and the high possibility of functional group modifications, which was beneficial for protein loadings.32 The response signals of the two analytes on their respective sensor were recorded simultaneously and the ratios of Aβf versus Aβo were calculated. As in the process of AD development, the selfaggregation of Aβ will become more intense from oligomers to fibrils, accompanied with a gradually decreased Aβo and an increased Aβf in the contents, the ratio between Aβf and Aβo could be therefore taken as the a possible quantitative index to evaluate the aggregation degree of Aβ at different stages. Upon evaluating the analytical figures of merit of this method, its applicability was also demonstrated by analyzing cerebrospinal fluid (CSF) and tissue (hippocampus, cortex and striatum) samples from AD rats for their Aβf / Aβo concentration ratios during the time course. To our knowledge, this was the first report of an electrochemical assay that was able to assess the level of Aβ aggregation associated with AD development quantitatively.

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Scheme 1. Schematic diagram showing the sensing of Aβ oligomers and fibrils in the two-channel electrochemical system.

EXPERIMENTAL Materials and Reagents: All the information could be found in the Supporting Information. Instruments: This section could be found in the Supporting Information. Preparation of CNT-PIL: The detailed information about the preparation of CNT-PIL was provided in the Supporting Information. Synthesis of NLC-A11-Th or NLC-OC-Th bioconjugate: The preparation of nanostructured lipid carrier (NLC) was performed according to a previous report33 with a modification, which was provided in Supporting Information. To prepare NLC-A11-Th or NLC-OC-Th bioconjugate, 1 mL saturated thionine (Th) solution was added to 5 mL NLC, mixed and stirred efficiently at 1500 r/min for 24 h at room temperature to allow the interaction between NLC and Th. The resulting solution was concentrated to 800 μL by centrifugation (8000 r/min, 15 min, 25°C), washed and resuspended in the incubation buffer to obtain the NLC-Th composite. Then, 3.3 uL of A11 or OC was added to the prepared NLC-Th composite and incubated for 4 h in a shaker (25°C, 1500 r/min). After centrifugation (8000 r/min, 15 min, 25°C) and washing, the product was re-suspended in 266 uL incubation buffer. The resulting bioconjugate was denoted as NLC-A11-Th or NLC-OC-Th. When not in use, the bioconjugate should be stored in 4°C. Fabrication of the respective electrochemical immunosensor for Aβ1-42 oligomer and Aβ1-42 fibril: Before fabrication, bare glassy carbon electrode (GC) was firstly polished with 0.05 μm alumina paste on a micro-cloth and then cleaned using acetone, ethanol and distilled water with ultrasonication for 10 min, respectively and dried at room temperature. Firstly, 5 uL CNT-PIL suspension in dimethyl formamide (DMF) (2.7 mg/mL) was applied to the bare GC and dried under infrared light for 30 ~ 40 min. The unbound compound was washed away with doubly distilled water. Then, 5 uL EDC/NHS was dropwise casted onto CNT-PIL/GC for activation in air for 1~2 h and excess EDC/NHS was rinsed using distilled water. Next, 5 uL A11 or OC solution (diluted the primary antibody solution by three times with an antidiluent) was introduced to the above electrode and allowed to dry at room temperature for 6 h. After washing with PBS and PBST for several times to eliminate the

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physically adsorbed antibody, 10 uL blocking solution was applied to eliminate the non-specific binding effects and block the remaining active sites on GC. The obtained electrode was defined as CNT-PIL/A11/GC or CNTPIL/OC/GC. After another washing cycle with PBS and PBST, the finished sensor was stored in 4°C when not in use. Then, 5 uL Aβ1-42 oligomer (Aβo) or fibril (Aβf) standard solution with a certain concentration, which was prepared by diluting their stock solutions (1 mg/mL) with PBS (pH 7.4), was dropped to the above electrode and cultured overnight in a moisture-saturated condition at 37°C. In this process, as the fibril commonly had a poor solubility especially at higher concentrations, to complete the specific recognition more efficiently on a solid interface, before dropping, the fibril solution or suspension should be piped gently with a pipette in the ice bath for at least 100 times without any foam to ensure the analyte was uniformly distributed on electrode surface. Then it was washed with PBS and PBST alternatively to remove unbound targets. This washing step was quite important as during the overnight culture at 37°C, part of the oligomer solution tend to transform into fibril on the electrode but would be recognized by A11, therefore, the formed fibrils could be washed off the electrode. The obtained two electrodes were named as CNTPIL/A11/Aβo/GC or CNT-PIL/OC/Aβf/GC. Finally, 5 uL of each of the prepared NLC-A11-Th or NLC-OC-Th bioconjugate was casted and allowed to interact with their corresponding antigens by incubating the electrode for 6 h. The electrodes were then washed with PBS and PBST alternatively for three cycles and dried at room temperature for 2 h. The obtained two electrodes were denoted as CNTPIL/A11/Aβo/NLC-A11-Th/GC and CNT-PIL/OC/Aβf/NLCOC-Th/GC. Construction of the two-channel electrochemical analytical system: The simultaneous electrochemical monitoring of Aβo and Aβf were carried out on a twochannel electrochemical analytical system. In this system, the Aβo immunosensor was placed at the position of channel 1, while the Aβf immunosensor was at channel 2. By immersing the two electrodes into artificial cerebrospinal fluid (aCSF) solution at the same time under N2 atmosphere, the respective current response was recorded by differential pulse voltammetry (DPV) in the potential range of 0 to -0.5 V. For real sample determinations, in order to eliminate the possible interferences from brain system, standard addition method was adopted, which was performed by firstly adding a standard solution of Aβo or Aβf with a certain concentration into CSF or tissue homogenates. Then, the mixture was applied onto CNT-PIL/A11 or CNT-PIL/OC modified sensors and cultured, followed by casting the NLCA11-Th or NLC-OC-Th bioconjugate onto sensors. By linear fitting of added concentrations and the detected current signals, the contents of Aβo and Aβf in CSF and brain tissues could be calculated. Induction and evaluation of AD model rats: All the detailed information for the animal experiments was provided in Supporting Information. Cell - level toxicity verification: This section was also available in the Supporting Information. Statistical analysis: All data in the whole experiments were expressed as mean ± SD. The data shown were obtained from at least three independent experiments. Differences between the rat groups were assessed by the one-way

ANOVA and Dunnett’s post hoc test. All the statistical analyses were conducted by SPSS 19.0 software (SPSS, Inc, Chicago, IL). Significant differences were represented as **P < 0.01 or ***P < 0.01.

RESULTS AND DISCUSSION Characterizations of the prepared CNT-PIL and NLC-A11Th bioconjugate: The most important in the fabrication of a biosensor was the successful immobilization of recognition substances, e.g., enzyme, antibody, aptamer, etc. CNT was considered a promising support material to deposit nanoparticles or macromolecules. However, to achieve a sensitive and stable biosensor, surface modification of CNT was commonly required. In this work, polymeric ionic liquid (PIL) was selected as a possible alternative to modify CNT to obtain a nanocomposite CNT-PIL. PIL has been proved to be effective stabilizer or modifier for the synthesis and functionalization of various nanomaterials, including CNT, which was endowed with improved conductivity, excellent hydrophilicity and positive charged.34 The successful synthesis of CNT-PIL was confirmed by Raman, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and thermal gravimetric analysis (TGA) (Figure S3). The prepared bioconjugates NLC-A11/OC-Th were also analyzed by transmission electron microscopy (TEM) measurements (Figure S4). Confirmation of sensor modification process: The two sensors for Aβo and Aβf were fabricated in a similar way, which were started from coating CNT-PIL as the substrate as to (1) immobilize antibody onto the blank sensors and (2) facilitate the electron transfer on the sensors. After that, the sensor fabrication was processed in a “sandwich” form, first was the coating of antibodies (A11 and OC) on CNT-PIL modified sensor, then was specific recognition of the sensor surface-confined antibodies to Aβ species (Aβo and Aβf), followed by the last dropping of NLC-A11/OC-Th bioconjugate. The electrochemical detection of Th reduction was used for quantitative analysis of Aβo and Aβf in aCSF solution (pH 7.4). The obtained two sensors were denoted as CNT-PIL/A11/Aβo/NLC-A11-Th and CNT-PIL/OC/Aβf/NLCOC-Th. To verify the sensor modification process, scanning electron microscopy (SEM), XPS and electrochemical measurements were adopted. As shown in Figure S5, compared with pure CNT (Figure S5A), after antibody was modified onto sensor surface, the tubular structure of CNT became vague, which was due to the dense coverage of multiple antibody layers (Figure S5B). With the further modification of Aβo, the protein layer covered on the surface of CNT became thicker, and basically the original tubular morphology of CNT could hardly be observed (Figure S5C). In the last step, when the sensor was modified with the NLCA11-Th bioconjugate, round spheres in the CNT network were observed, which was attributed to the appearance of NLC in the film, indicating that the NLC-A11-Th bioconjugate was also successfully bounded to the sensor (Figure S5D). Meanwhile, it was observed from Figure S5D that the size of NLC-A11-Th bioconjugate was about 340 nm, which was bigger than that from TEM characterization (205 nm, Figure S4B). The reason for this discrepancy was possibly that when SEM was tested, the NLC-A11-Th bioconjugate was dropped onto the solid sensor surface and incubated for 6h to interact with antigen. During this process, part of the antibody would

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be released from the bioconjugate and bind with antigen. As a result, NLC lost the protection from protein encapsulation and became instable again. Therefore, the NLC particles gradually fused together, resulting in a larger particle size. As displayed in Figure 1A, a predominant C 1s peak at ~284.8 eV was observed on the four surfaces, which belonged to the standard reference carbon.35 Following immobilizations of A11 and Aβo, an additional peak at ~286.1 eV appeared, which corresponded to C-N bond, due to the linking between carboxylic acid groups in PIL and amine groups in A11.36 Upon the modification of NLC-A11-Th bioconjugate, a new peak at 289.1 eV appeared, which was assigned to O–C=O bonds.37 For N 1s (Figure 1B), a peak at ~399.5 eV after the immobilization of A11 onto CNT-PIL was presented, which belonged to the N-C bonding.38 The peak at ~ 402.1 eV on the surface of CNT-PIL could be ascribed to the tertiary nitrogen (N-(C)3) in the skeleton of PIL.39 After the modification of NLC-A11-Th, N 1s was separated into two peaks at ~399.1 and ~402.6 eV, which could be attributed to sp2-hybridized nitrogen (C=N–C) and terminal amino functions (C–N–H) due to the presence of Th.40 Moreover, the characteristic peak of P 2p at ~133.1 eV only appeared when the bioconjugate was coated on the sensor surface (Figure 1C). Additionally, the ratios of atomic percent of C 1s and N 1s were determined, which were 42.54, 11.90, 10.92, 28.73 for the four surfaces, indicating the state changes of sensor surfaces due to the modification process. Electrochemical behaviours of K3Fe(CN)6 was proved a valid tool for evaluating the kinetic barrier of the interface.41 Figure 1D illustrated the typical cyclic voltammetry (CV) curves obtained on different sensors in 1 mM K3Fe(CN)6 solution. A pair of well-defined redox peak assigned to the redox couple of Fe3+/Fe2+ was obtained on the four sensors. Compared with bare sensor (curve a), both anodic and cathodic currents of curve b increased on CNT-PIL. Additionally, the peak-topeak potential (△Ep) was calculated to be 0.88 V for bare sensor and 0.82 V for CNT-PIL. The enhanced peak currents, combined with a decreased △Ep value, indicated an improved electron transfer rate on CNT-PIL. However, when A11 and Aβo were immobilized onto CNT-PIL/GC, the peak currents decreased significantly with enlarged △Ep values (0.93 and 0.94 V for curve c and d, respectively), which was caused by the intrinsically poor conductivity of proteins. The current signal continued to drop off after NLC-A11-Th bioconjugate was casted and the △Ep increased to 0.112 V. Although Th itself in the bioconjugate was conductive, the huge bulk of NLC and massive A11 loadings hindered the electron transfer of Fe3+/Fe2+ on the sensor, thereby the current was still not recovered and resulted in a poorer redox behavior. These evidences jointly suggested that the two sensors have been fabricated successfully.

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Figure 1. XPS spectra of (A) C 1s, (B) N 1s, (C) P 2p on CNTPIL, CNT-PIL/A11, CNT-PIL/A11/Aβo and CNTPIL/A11/Aβo/NLC-A11-Th modified surfaces. (D) The typical CV curves of bare GC (a), CNT-PIL (b), CNT-PIL/A11 (c), CNT-PIL/A11/Aβo (d) and CNT-PIL/A11/Aβo/NLC-A11-Th (e) modified sensor in 0.1M KCl solution containing 1 mM K3Fe(CN)6. Simultaneous determination of Aβo and Aβf in the constructed two-channel system: Figure 2 showed the DPV responses of Aβo and Aβf mixtures (1 ng/mL for each of them) on their corresponding immunosensor in a N2saturated aCSF solution. It could be seen that both Aβo and Aβf exhibited a rather similar electrochemical behavior on the surfaces of their respective sensor in one system, that was, around -0.3 V, a clear reduction peak ascribed to the reduction of Th appeared. We could make a distinction between the two analysts from their current responses that, the determined current of Aβo was higher than that of Aβf for the same concentration. This evidence clarified that the simultaneous and specific identification and determination of Aβo and Aβf in the same electrochemical system was possible and feasible. A control experiment was parallelly performed to test the specificity of the two sensors toward their targets, that whether the A11 containing sensor could bind fibril and OC containing sensor could bind oligomer. As indicated in Figure 2A, neither A11 nor OC containing sensors could produce remarkable responses toward fibril and oligomer (dashed lines), confirming a high specificity of the two sensors toward their respective analyte. Moreover, to illustrate the accuracy of this analytical strategy, we compared the DPV responses of Aβo and Aβf at their immunosensors determined in separate and simultaneous manners. As indicated in Figure 2B and 2C, the DPV plots of both Aβo and Aβf recorded in the one- and two- channels were basically the same, indicating that there was no crosstalking effect between the two analytes when electrochemical monitoring was performed in one system. After incubating CNT-PIL/A11/Aβo/GC in the bioconjugate solution, the bioconjugate could bind to Aβo due to the specific recognition between A11 and Aβo. In order to demonstrate the specific interaction between A11 and Aβo and the enhanced sensitivity of this strategy by NLC, three sensors were parallelly fabricated that coating A11-Th, NLCTh and NLC-A11-Th films onto CNT-PIL/A11/Aβo/GC, respectively and their DPV responses were recorded and

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compared. As shown in Figure 2D, the current signals obtained on A11-Th and NLC-Th films were much smaller than that on NLC-A11-Th. On one hand, the lack of A11 in the NLC-Th composite resulted in the incapability of recognition and thus the failure in the immobilization onto CNTPIL/A11/Aβo/GC surface. On the other hand, comparison between NLC-Th and NLC-A11-Th films indicated the amplification effect by NLC due to its special structure that provided a useful platform for the massive loading of proteins. Also, the nature-derived property of NLC was beneficial for maintaining the bioactivity of antibodies. This result illustrated that this immune strategy for the determination of Aβo and Aβf by virtue of NLC and Th was feasible and reliable.

Figure 3. Differential pulse voltammetry (DPV) responses toward different concentrations of Aβo and Aβf (A and C) and the corresponding linear plots (B and D) obtained on CNT-PIL/A11/Aβo/NLC-A11-Th and CNT-PIL/OC/Aβf/NLCOC-Th sensors. The tested concentrations of Aβo and Aβf were 0.2 (a), 0.4 (b), 1.0 (c), 2.0 (d), 10.0 (e), 20.0 (f), 40 .0 (g) ng/mL. Data in B and D were expressed as mean ± SD (n =3).

Figure 2. (A) DPV responses of 1 ng/mL Aβo and Aβf on their respective immunosensor in N2-saturated 0.1 M aCSF solution in the two-channel electrochemical system. The two dashed lines represented DPV responses of Aβo (red) and Aβf (black) on OC and A11 modified sensors, respectively. (B) and (C) were DPV responses of 1 ng/mL Aβo and Aβf on their respective immunosensor detected in one- and two-channel mode. (D) Comparison of DPV responses toward 1 ng/mL Aβo using A11-Th, NLC-Th and NLC-A11-Th as the bioconjugate to recognize Aβo. Under the optimized conditions (see section 2.4 in SI, Figure S6), good linear relationships between the recorded currents and the logarithm of Aβo or Aβf concentrations in the range of 0.2 to 40 ng/mL were established for the two sensors (Figure 3). The regression equations were Ip (μA) = 18.99 lgC (ng/mL) + 36.44 for Aβo and Ip (μA) = 10.37 lgC (ng/mL) + 33.37 for Aβf, respectively, with the linear correlation coefficients of 0.994 and 0.995. The detection limits of the two as-prepared sensors were 0.01 and 0.02 ng/mL for Aβo and Aβf, at the ratio of signal to noise of 3. We compared the linear range and detection limit among our work and other reported literatures (Table S2), which illustrated that the performance of our proposed strategy provided higher sensitivity and wider linear range than other biosensors or even far superior to some enzyme biosensors.

In order to apply this modified electrode into determination of Aβo and Aβf in rat brain, the selectivity of this assay was examined. Potential substances that might interfere with the detection of Aβo and Aβf included metal ions, amino acids, other isoforms of Aβ, all of which coexisted in the same cerebral system. In the interference experiments, we measured the DPV signals of these biological species separately in the same way as Aβo and Aβf or by observing the signal changes with mixtures of these species with Aβo and Aβf. As could be observed from Figure S7, compared with the response of Aβo, rather weak currents were obtained in the presence of metal ions and amino acids (P ＜ 0.001), which was caused by the absence of specific recognition elements for these species, resulting in the incapability to bind onto electrodes. Apart from these species, we also inspected the effects of Aβ monomers, different sequences of Aβo and Aβf on the measurement of targets. It was clearly shown that responses from Aβ1-42 monomer, aggregated forms of Aβ1-11 and Aβ1-16 were comparatively weak with respect to our targets (P ＜ 0.001). Moreover, the produced electrochemical signals of the mixtures were almost the same as that of the Aβo or Aβf alone without significant differences. However, we noted that oligomeric and fibrillar Aβ1-40 also produced similar current signals to those of Aβ1-42. This observation was consistent with previous reports that A11 and OC were specific to sequences of Aβ containing 40 and 42 amino acids.20 In general, this immune two-channel system exhibited acceptable selectivity toward Aβo and Aβf over coexisting biologically relevant species or other Aβ isoforms, and could be applied for biosensing in real samples. For repeatability and reproducibility evaluations, six electrodes with the same concentration of Aβo and Aβf were fabricated in parallel on the same day and consecutively for six days, respectively, and the corresponding DPV responses were recorded and compared. The two relative standard deviations (RSDs) were calculated to be 3.8% and 2.7% for

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Aβo, 3.5% and 6.4% for Aβf (n = 6), indicating acceptable repeatability and reproducibility for the simultaneous determination of Aβo and Aβf. In addition, the two sensors also displayed reasonable stabilities for their substrates, in which less than 10% declines of the original Aβo and Aβf responses were obtained after sensors were stored in 4°C for one week. These combined results illustrated the reliability of the two-channel system for the simultaneous determination of Aβo and Aβf, which met the requirement of monitoring Aβ aggregates in biological samples.

Figure 4. (A) Incubation time-dependent generations of Aβo and Aβf at 37°C measured in the two-channel system. (B) Derived calibration curve corresponding to the [Aβf] / [Aβo] at different incubation time. The concentration of incubated Aβ monomer was 1 ng/mL. Evaluation of Aβ aggregation: Based on the excellent performance of the two-channel system, we next investigated the aggregation process of Aβ quantitatively by this electrochemical approach. Figure 4A showed the representative time-resolved DPV responses of 1 ng/mL Aβ standard solution incubated in a 37°C water bath for one to seven days. As indicated, as time went by, the corresponding peak currents of Aβo decreased gradually and at the same time, we observed a progressive increase in the signals of Aβf, indicating that along with the prolong of incubation time, the extent of Aβ aggregation became more and more severe, during which, more oligomers have transformed to fibril forms. Noticeably, in this process, although the general tendency of the variability for Aβo and Aβf was consistent, the change degree at a certain stage was different from each other. The most significant change of the obtained current responses occurred on the 4th day for Aβo and the 3rd day for Aβf. This discrepancy might be caused by the different sensing abilities of the two kind sensors. Therefore, to overcome this inconformity, we proposed that the degree of Aβ1-42 aggregation could be judged by the calculated ratios of peak currents between fibrils and oligomers, which could eliminate the possible environmental effects on the single detected signal and thus results would be more accurate. It was found that the ratio kept increasing as incubated time duration (Figure 4B). As the self-aggregation and oligomerization of Aβ were associated with the pathology of AD, we next evaluated the Aβ-associated neurotoxicity during the conversion of oligomers into fibril aggregates by incubating mouse neuroblastoma cells (N2a), with the aggregated Aβ proteins for 72 h (Figure S8). In general, the shorter of the incubation time was, the lower of the cell survival rate would be. This was due to the formation of amyloid fibrils which were comparatively less toxic than oligomers. These findings also suggested that a decrease in the concentration of oligomers correlated with a decrease in Aβ-associated toxicity, which was consistent with the results

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in Figure 4. It could be seen from the results that the survival rates of cells after treatment with Aβ aggregates were comparatively low, which implied that the aggregated forms of Aβ had a great toxicity to nerve cells and a certain influence on the whole nervous system. To convince the electrochemical results, another two experiments, TEM and thioflavin T (ThT) fluorescence assay were also conducted (Figure 5). The obtained TEM images clearly probed the gradual lengthening and replacement of oligomers with fibrils as the incubation time prolonged. After incubation Aβ monomer for one day (day 1), the image showed a mostly oligomeric form and no mature fibrils (Figure 5A). After day 2, the Aβ sample exhibited a sharp increase in the number of longer mature fibrils in a meshed network (Figure 5B-D). Until day 7, nearly no short fibrils could be found on the entire TEM grid which was indicative of full fibrillation (Figure 5E). This conformation-associated aggregation process of Aβ was further characterized by ThT fluorescence assay. As shown in Figure 5F, the characteristic ThT fluorescence spectra of Aβf with an emission peak at 500 nm were obtained with the excitation wavelength at 435 nm. The fluorescence intensity of Aβf increased with incubation duration, which reflected a similar tendency to that of electrochemical detection assay and TEM results that more fibrils have been formed with duration of incubation time.

Figure 5. Time course aggregation of Aβ monitored by TEM (A-E) and ThT fluorescence (F). The scale bars represented 200 nm. The concentration of incubated Aβ monomer was 20 ng/mL. Aβ species were formed by incubating 20 ng/mL Aβ monomer solution in a 37°C water bath for 1 to 7 days. Demonstration of Aβ aggregation process in AD rat brain: We next applied this strategy to track Aβ aggregation in AD rat brains. Contents of Aβo and Aβf in hippocampus, prefrontal cortex and striatum tissues from normal, control and AD rats were measured by standard addition method and the ratios between Aβf and Aβo were calculated. Meanwhile, to illustrate the dynamic progress of Aβ aggregation, the changes of aggregate contents and this ratio at different time courses of AD (one, two, three, four and five weeks after modeling completion) were also evaluated. As expected, the contents of Aβo in CSF and tissues from AD rats were comparatively higher than Aβf at one week (Figure 6A and Table S4). Moreover, the quantifications clearly indicated that aggregation of Aβ occurred most seriously in CSF in comparison with other three brain tissues, as reflected by the highest values of Aβo and Aβf, respectively, followed by the hippocampus, cortex and striatum. This finding was

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highly consistent with our previous report that the reduction in soluble Aβ monomer levels was greatest in CSF.31,42 The time course results in Table S5 and Figure 6B demonstrated that at very early stage, the calculated [Aβf]/[Aβo] value was 0.054, indicating that most of Aβ aggregates was in the form of oligomer. Next, at two weeks, both of Aβf and Aβo contents ascended whereas the increment of Aβo was greater than that of Aβf and therefore, a decline in the ratio was witnessed. After this time point, the content of Aβo decreased along with the on-going rising of Aβf contents, suggesting that more and more oligomers aggregated into longer fibrils and therefore we observed a gradually elevated ratio between Aβf and Aβo. The obtained electrochemical results could be confirmed by ThT assay and representative TEM images (Figure S10), which also showed a similar tendency to those of Aβf and Aβo standard solutions. These findings verified that consistent with the AD-Aβ association theory, the progress of AD always accompanied by the selfaggregation process of Aβ monomer, which will firstly selfassemble into larger oligomers at early stage of this disease and eventually, this peptide will mainly form the elongated fibrils that were observed in late-stage AD rats. These obtained results also excellently correlated with our previous data for tracking the dynamic aggregation of Aβ and further confirmed that this quantitative strategy that using the ratio between the two common Aβ aggregates could be reliably applied for the real-time evaluation of AD pathogenesis.

low amount of aggregates. Alternatively, the aggregation extent of Aβ standard solution or CSF or tissue homogenates from AD rats could be reliably evaluated by calculating the ratio of Aβf versus Aβo at different time stages, which may provide a possible index for clinical diagnosis of AD patients.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. It included additional experimental details; characterizations of IL-COOH, CNT-PIL and NLC-A11-Th bioconjugate; SEM images for the sensor modification process; optimization process; selectivity; cell viability evaluation; verification of AD rats; TEM and ThT fluorescence assay of hippocampus from AD rats.

AUTHOR INFORMATION Corresponding Author *Phone/Fax: +86 516 8326-2009, e-mail: [email protected] *Phone/Fax: +86 516 8326-2138, e-mail: [email protected]

Author Contributions ‡ These

Notes

authors contributed to this work equally.

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (no. 21675137), Natural Science Foundation of Jiangsu Province (no. BK20161170), China Postdoctoral Science Special Foundation (no. 2016T90504), Natural Science Foundation of Jiangsu Higher Education Institute of China (no. 17KJA350004), Program for Distinguished Talents of Six Domains in Jiangsu Province (no. 2016-SWXY-060) and Jiangsu “333” project of cultivation of high-level talents.

REFERENCES

Figure 6. (A) Aβo and Aβf contents in CSF and fixed brain tissues of AD rats at one week after modeling completion; (B) Aβo and Aβf contents in the hippo of AD rats at one, two, three, four, five weeks after modeling completion. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CSF (A) and one week (B).

CONCLUSIONS Overall, a novel strategy that was able to quantify the simultaneous changes in the level of Aβ oligomers and fibrils in AD brains in one system was designed. In this system, two independent sensors that were specific to the oligomeric and fibrillar forms of Aβ were sensitive enough to quantify the

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