In Situ Monitoring the Aggregation Dynamics of Amyloid-Î² Protein

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In situ monitoring the aggregation dynamics of Amyloid-# protein A#42 in physiological media via Raman-based frequency shift method Wenfeng Zhu, Yibing Wang, Dan Xie, Linxiu Cheng, Ping Wang, Qingdao Zeng, Min Li, and Yuliang Zhao ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00257 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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In situ monitoring the aggregation dynamics of Amyloid-β protein Aβ42 in physiological media via Raman-based frequency shift method Wenfeng Zhu†,ǁ,c, Yibing Wang‡,ǁ, Dan Xie†, Linxiu Cheng†,∆ , Ping Wang‡, Qingdao Zeng∆,*, Min Li †,*, Yuliang Zhao†,∏, † CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, 19B Yuquan Road, Shijingshan District, Beijing 100049, China. ‡ State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology (ECUST), Shanghai 200237, People's Republic of China. ∏

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience and Technology (NCNST), Beijing 100190, China ∆ CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology (NCNST), Beijing 100190, China


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c Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China KEYWORDS: frequency shift biosensing, SERS, Aβ42, Alzheimer’s disease, aggregation process

ABSTRACT: Amyloid-β protein (Aβ) is a major biomarker candidate for diagnosis of Alzheimer’s disease (AD). It is known that the core segment of Aβ42 tends to aggregate into neurotoxic soluble oligomeric species and finally into fibrillar structures associated with AD, however, much remains to be learnt about the conformational changes and dynamic aggregation processes of Aβ protein in solution. Herein we exploit the selectivity of affinity peptides, singled out by biopanning a phage display library, to recognize and capture Aβ42 and its fibers. The sensitivity of surface-enhanced Raman spectroscopy (SERS) to subtle electronic changes of a Raman reporter upon Aβ42 binding, that is, the frequency shift SERS assay, is employed to develop a reliable sensor for both in situ Aβ42 aggregation monitoring and Aβ42 monomers, fibers detection. Atomic force microscope (AFM) imaging is used to investigate the dynamic aggregation processes of Aβ42 on mica and confirms the conclusions of the SERS studies. Finally sensing of Aβ42 and its fibers in fetal bovine serum (FBS) solution is shown to have a limit of detection of ~10-9 mol/L.

1. Introduction


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Alzheimer's disease (AD), first reported in 1906 and manifested as progressive cognitive decline, is the most common cause of dementia. The number of people living with AD is projected to be 13.5 million by 2050.1 Clinical evidence indicates that the neuropathology usually starts more than 10 years before AD manifests clinically.2-4 The differences between typical age-related cognitive changes and indications of AD can be subtle and easily missed. Although scientists have determined the high-definition structure of AD-related proteins by using cryo-electron microscopy technology5, 6, currently there is no effective means of diagnosis and treatment of AD. AD has become a public health problem affecting the global aging population. In recent years, great efforts have been made to develop and validate AD biomarkers for clinical diagnosis, including those in blood and cerebrospinal ﬂuid.3, 7 Accumulating evidence suggests that Amyloid-β protein (Aβ), present in two significant isoforms Aβ40 and Aβ42, is associated with AD. The core segment of Aβ42 particularly aggregates into neurotoxic soluble oligomeric species and finally into fibrillar structures.8,

9

This aberrant oligomerization and

structure misfolding leads to senile plaques and deposits in the brain, which then lead to neuronal dysfunction and cell death. Since Aβ42 has a higher tendency to fibrose than Aβ40, studying the dynamic aggregation behavior of Aβ42 in aqueous solution can give insight into AD genesis and development, and most relevant here, offer possible avenues for early AD diagnoses. Much effort has been devoted to the study of Aβ aggregation processes. For example, threedimensional solution structures of both Aβ40 and Aβ42 were determined by NMR spectroscopy in aqueous sodium dodecyl sulfate (SDS) micelles.10,

11

Voller, Teplow, and co-workers

quantified the formation of pentamer/hexamer units and beaded superstructures which are similar to early protofibrils of Aβ42.12 Other methods such as electron paramagnetic resonance (EPR)


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spectroscopy 13 and magnetic resonance imaging (MRI) 14 were also applied to structural studies of Aβ42. There are several reports on the detection of oligomers or aggregation of amyloid peptide by peptide based probes15, conjugated polymer probes16, nanoparticles17, selective luminescent probes18 and other small molecule fluorescent systems19-21. However, the development of blood-based assays for in situ monitoring the aggregation process of Aβ42 is in its relative infancy though some fluorescent probes can discriminate Amyloids22. In addition, in situ studies on Aβ42 aggregation behavior and blood-based Aβ42 detection are essential for revealing the pathogenesis and clinical diagnosis of AD. Surface-enhanced Raman scattering (SERS) spectroscopy is a valuable tool for qualitative and quantitative analysis of chemical identity and structure. SERS has been widely employed in biology science for the detection and analysis of nucleic acids, proteins, even cells and tissues.23 It can be also used to monitor H-Bonding reactions between molecules,24, 25 immunoreactions between antibodies and antigens,26 hybridization of oligonucleotides,27, 28 and specifically £amyloid

29-31

. The ‘fingerprint’ spectral frequencies of a Raman reporter can be highly sensitive

to the dielectric environment, solution pH, and molecular weight of interacting species. This suggests frequency shift SERS assays might be a promising tool for the detection and monitoring of the aggregation process of Aβ42 in solution. Such assays require the selective recognition and capture of the Aβ protein and its aggregates at a metallic nanoparticle surface. Antibodies can provide the most direct recognition and have been used in the construction of several biosensors for Aβ detection.32 However when decorated onto nanoparticles, large native antibodies can lose their structure and function due to the interfacial action.33, 34 Although some affibodies (affinity antibodies with smaller size) have been


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developed for Aβ,35, 36 peptides consisting of the smallest active domain of the protein may be a superior alternative for biosensor construction as they offer higher surface density and structural fidelity. There are some reports on the use of affinity peptides to construct biosensors for the detection of cells, proteins or even serum Aβ37, 38 Another advantage of replacing natural antibodies with affinity peptides is that they can be efficiently produced using in vitro selection procedures and microbes. These short chain, highaffinity peptides can be selected by biopanning of phage display libraries,39, 40 then the selected peptides are further utilized as the optimal ligand for target capture. So far, many selective affinity peptides targeted at cells,41 bacterium,42-44 proteins,45, 46 and organic small molecules

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have been developed in this way and used to realize biosensors48, 49 Affinity peptides selected by biopanning against the different forms of Aβ (fibers, oligomers and monomers) have the potential to be used as recognition ligands for biosensors to detect the process of Aβ aggregation in physiological media. Herein we exploit affinity peptides obtained by biopanning a phage display library to recognize and bind Aβ42 monomers and fibers. The binding efficiency is characterized using SERS spectroscopy, and studies over time allow dynamic aggregation processes of Aβ42 to be monitored, with corroborating results from AFM imaging. Finally, sensing of Aβ42 monomers and fibers in physiological media is demonstrated with a moderate limit of detection. 2. Experimental Section 2.1 Materials


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The phage library that displays 12-mer peptides (Ph.D.-12 Phage Display Library Kit) was purchased from New England Biolabs, Inc (NEB, Ipswich, MA, USA). Silver nitrate (AgNO3), glutaraldehyde (25% aq. solution), ethylene glycol, (3-mercaptopropyl) trimethoxysilane (95%) and acetonitrile were obtained from Alfa Aesar (Shanghai, China). Ethanolamine and 1,1,1,3,3,3Hexafluoro-2-propanol (99%) were purchased from Sigma-Aldrich (Shanghai, China). Sodium hydroxide (NaOH), ethanol, sulfuric acid (98%), toluene and ammonium hydroxide solution (NH3·H2O, 28–30%) were bought from Beijing Chemical Reagent Factory (Beijing, China). Biotin-β-Amyloid (1-42), human, was custom-synthesized by China Peptide Co., Ltd (Shanghai, China). Tris-HCl buffer (0.05 mol/L, pH 7.4, Sterilized) was obtained from SunBio Science (Nanjing, China). Fetal bovine serum (FBS) was purchased from Gibco (Australia). DSNB (5,5’dithiobis(succinimidyl-2-nitrobenzoate) was synthesized according to the previously reported method.50 2.2 Biopanning of phage-display random peptide library Biopanning experiments were carried out according to the instruction manual of the phage display library kit. For the biopanning of Aβ42 monomer binding peptide, biotinylated Aβ42 was introduced to bind with streptavidin coated on the surface of an individual cell culture plate. 10 µL M13 phage display 12-mer peptide library (containing about 1011 phage and 109 different peptide sequences) was pre-mixed with 2 µmol/L biotin-Aβ42 in 1.5 mL TBST (50 mmol/L TrisHCl buffer, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) for 60 min. This pre-complex suspension was poured into the streptavidin coated plate, and incubated for 10 min. Before washing, 0.1 mmol/L biotin was added and incubated for 5 min to displace any streptavidinbinding phage. The solution in the plate was then poured off and the plate washed with TBST six times. The concentrations of Tween-20 in the TBST washing buffer were 0.1%, 0.3% and 0.5%


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for the first, second and third rounds of biopanning, respectively. Bound phage was eluted by 1 mL of glycine elution buffer (0.2 mol/L glycine-HCl, pH 2.2, 1 mg/mL BSA) for 10 minutes incubation at room temperature, then the eluted suspension was neutralized by 150 µL Tris-HCl buffer (1 mol/L, pH 9.1). This eluted phage was amplified in host E. coli (ER2738) for the next round of biopanning. After three rounds of biopanning, individual phage clones were separated according to the instruction manual. The ssDNA of the phage clones was extract using a biomiga M13 isolation Kit (Biomiga, Inc. San Diego, CA.), and sequenced (Shanghai RuiDi Biological Technology Co., Ltd). For the biopanning of the Aβ42 fiber binding peptide, Aβ42 was first dissolved in 1,1,1,3,3,3Hexafluoro-2-propanol to induce aggregation, and an appropriate amount of solution was then transferred to a microcentrifuge tube for solvent evaporation. These peptide deposits were dissolved in TBST (50 mmol/L Tris-HCl buffer, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) to a final concentration of 50 µmol/L. The peptide solutions were incubated at 37 °C for two days to obtain the Aβ fiber. 10 µL M13 phage display 12-mer peptide library was added into βAmyloid fiber suspension, then this suspension was rocked and incubated for 30 minutes at room temperature. After incubation, the fiber and binding phage were separated from unbound phages by spinning at 12,000g in an Eppendorf 5418R centrifuge for 10 min. The deposits were then washed with TBST six times (the concentration of Tween-20 for the first, second, third and the fourth round were 0.05%, 0.1%, 0.3% and 0.5%, respectively). Then, the suspension was transferred to another fresh microcentrifuge tube. After spinning, phage which remained bound to the fiber was eluted by 1 mL of glycine elution buffer (0.2 mol/L glycine-HCl, pH 2.2, 1 mg/mL BSA) for 10 minutes incubation at room temperature followed by centrifuging at 12,000g for 1 min. The supernatant was transferred to another fresh microcentrifuge tube and


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neutralized with 150 µL Tris-HCl buffer (1 mol/L, pH 9.1). The eluted phage was then amplified for the next round of biopanning. After four rounds of biopanning, individual phage clones were separated and the ssDNA of phage clones extracted and sequenced. 2.3 Silver Nanoparticle Films (Ag NFs) Preparation for SERS measurements. Glass slides of 0.8 × 0.9 cm2 were ultrasonicated in ethanol for 10 min, and thoroughly cleaned with piranha solution at 95 °C for 40 min (H2SO4:H2O2 = 3:1, v/v). After rinsing with water and drying under N2 flow, the cleaned glass slides were modified in a 2% 3mercaptopropyl-triethoxysilane solution in toluene overnight. Silanized glass slides were obtained by rinsing with toluene and ethanol respectively, and subsequently dried under N2 flow for further use. To a 50 mL solution silver nitrate solution (2.6 mmol/L), 135 µL 10% NaOH was slowly added. Following that, 10% ammonia was added dropwise, until the pale brown mixture became transparent again. The obtained solution was cooled in an ice-water bath for 10 min and 300 µL 25% glutaraldehyde was added. After stirring it for 10 s, the solution was placed in a 90 °C water bath and the silanized substrate suspended within the solution immediately. After 4 min of silver nanoparticle growth on the substrate an optically dense film formed (the Ag NF). The Ag NF was then placed in pre-cooled ethylene glycol immediately to stop the reaction. Ag NFs were ultrasonically cleaned for 20 s to remove non-specific absorbed silver nanoparticle and stored in ethylene glycol at 4 °C before use. 2.4 Probe modification and Sensing Ag NFs were immersed in a 1 mol/L solution of the Raman reporter DSNB in acetonitrile at room temperature for 4 h to form a dense surface layer by Ag-S binding. Then the substrate was incubated with 0.01 mg/mL solution of the peptides for monomer or fiber capture (probe P1 and


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P2, see below) for 2 h to obtain the probe-modified AgNFs. Because peptides were covalently immobilized on DSNB/AgNFs, single monolayer of peptide could be obtained after washing away the ones that were physically adsorbed on surface. Therefore, the aggregation of these immobilized peptides is not possible to occur, thus the affinity peptides have no pronounced contribution to the frequency shift upon Aβ42 sensing. The substrates were then immersed in 1 mmol/mL ethanolamine for 30 min at room temperature to block the unreacted DSNB, followed by rinsing with Tris-HCl buffer. Probe modified AgNFs were immersed in Tris-HCl buffer containing 4.6 × 10−6 mol/L Aβ42 and the SERS spectra were in situ recorded on a DXR Smart Raman spectrometer (Thermo Fisher, 780 nm,

40 mW, 10 µm diameter focal spot laser

excitation, 15s integration time, room temperature). The Si (111) wafer was used to calibrate the Raman peak after each sample was measured. To evaluate sensor performance, SERS spectra were collected after the probe modified AgNFs were immersed in Aβ42 solutions with concentrations ranging from 4.6 × 10−6 mol/L to 4.6 × 10−10 mol/L. Incubation times were 60 min for P1-modified and 300 min for P2-modified substrates, respectively. The absolute shift in the Raman peak (|∆ Raman shift|) at 1334 cm-1 due to the nitro stretch of DSNB was used to assess binding efficiency for both Aβ42 monomer and fiber detection. To test the selectivity of the capture peptides, the same experiments were repeated in fetal bovine serum instead of Tris-HCl buffer, keeping all other conditions identical. 2.5 Control experiment P2-modified AgNFs were prepared as described above and stocked in Tris-HCl solution for further use. Aβ42 solution with 4.6 × 10−6 mol/L concentration was stayed at room temperature


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for 3h, 4h and 5 h, respectively, before Aβ42 solution was incubated with P2-modified AgNFs for another 1h. The Raman shift at 1334 cm-1 was recorded. 2.6 Atomic Force Microscope (AFM) characterization 20 µL of 4.6 × 10−6 mol/L solutions of Aβ42 in Tris-HCl buffer incubated for different amounts of time were deposited on freshly-cleaved mica substrates. The mica substrates were then thoroughly washed with ultrapure water and dried in air. AFM images were collected with a Nanoscope IIIa microscope system (Bruker, USA) in tapping mode. 3. Results and Discussion 3.1. Peptide sequence analysis and possible affinity mechanism Biopanning was employed to obtain affinity peptides for recognition of two stable forms of Aβ42, monomers and fibers. The biopanning procedure varied slightly in each case (see Methods). After three rounds of biopanning against the monomer and four rounds of biopanning against the fiber, affinity phage clones were enriched with the display peptide sequences shown in Table 1. For the Aβ42 monomer, two peptides sequences were obtained with peptide P1 showing the highest frequency while being displayed in the obtained affinity phage clones (6/7). The same peptide was also found in affinity selection for the fiber (N4) suggesting that peptide P1 may have affinity for the terminal region of the Aβ42 fiber. For Aβ42 fibers, three peptides sequences were obtained, with peptide P2 showing the highest frequency (11/13). The molecular structures of the monomer-binding peptide P1 and fiberbinding peptide P2 are shown in Figure 1 and feature different sequence characteristics. To analyze the binding affinity of selected monomer-binding peptide and targeted monomer, we


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compared the sequences of peptide P1 with Aβ42. The P1 peptide has three aromatic amino acid residues (H, His), which may have affinity for the sequence YEVHHQKLVFF of Aβ42 via π-π interactions between aromatic side groups. The Pro residue near the three His residues provides a rigid and stable structure for binding. In contrast, peptide P2 has amino acid residues that can take part in hydrogen bonding and is very flexible, not featuring Pro (P) or aromatic amino acid residues (H, F and Y). These amino acid residues with flexible side groups could specifically bind at the hydrophilic surface of Aβ42 fibers according to the recently reported work.5 Taking inspiration from these native affinity peptides, sequences with improved affinity could be obtained by molecular simulation and testing. This will be subject to the future work. Most importantly here, the different affinity peptides for Aβ42 monomers and fibers provide the possibility to develop a peptide-based sensor for recognizing Aβ42 and monitoring its dynamic aggregation process in physiological media. Table 1. Sequences of peptides displayed in phage clones Monomer affinity Peptide

Sequence

Frequency a)

N1 (P1)

GNNPLHVHHDKR

6/7

N2

DYHDPSLPTLRK

1/7

Fiber affinity peptide

Sequence

Frequency

N3 (P2)

QVNGLGERSQQM

11/13

N4 (P1)

GNNPLHVHHDKR

1/13

N5

HMYYPGGDGRFA

1/13


11


a)

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The frequency is the proportion of displayed phage clones corresponding peptide in all the

selected phage clones.

Figure 1. Molecular structures of the affinity peptides P1 and P2 for Aβ42 monomer and fiber, respectively, as obtained from biopanning. 3.2. Detection of and monitoring Aβ42 with affinity peptide We developed a frequency shift surface-enhanced Raman scattering (FS-SERS) assay for detection

of

the

Aβ42

monomer

and

fiber.

In

brief,

a

Raman

reporter

5,5’-

dithiobis(succinimidyl-2-nitrobenzoate), (DNSB) with a high cross-section for its nitro stretch at 1334 cm-1 is attached to a plasmonically active silver nanoparticle film (AgNF). The affinity peptides are then covalently bound to the reporter to give final probe for Aβ42 monomer or fiber target capture. Binding of these species leads to changes in the chemical structure of the reporter, shifting the frequency of the nitro stretch to higher energy, as quantified by SERS spectroscopy.


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Figure 2a shows a SEM image of the as-prepared AgNF used for SERS measurements on a glass substrate. AgNPs densely decorated the glass slide, having an average diameter of ~ 40 nm. These films have excellent sensitivity for SERS measurements with enhancement factors ca. 107.42 The process of AgNF modification with DSNB and affinity peptides P1 and P2 for Aβ42 monomer/fiber capture respectively is shown in Figure 2b (see also Methods for all details). DSNB is bound to the AgNF by Ag-S interactions while P1 or P2 are then covalently bound to DSNB by activated carboxylic coupling to give the final AgNF/DSNB/affinity peptide capture probe.

Figure 2. (a) SEM image of a typical silver nanoparticle film (AgNF) used in SERS measurements. (b) Schematic of AgNF modification with DSNB and affinity peptides P1/P2, and Aβ42 monomer/Aβ42 fiber capture, respectively.

The optimal conditions for sensor function including probe surface density, incubation temperature and incubation time were addressed first. It is reasonable that higher density of the affinity peptides on surface could lead to larger value of |∆ Raman shift| which is vital to the chip


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sensing performance. Different surface densities of the peptides on the sensor substrates were achieved by incubating the AgNF/DNSB substrate in different concentrations of affinity peptide. The substrates were then incubated with 4.6 × 10-6 mol/L Aβ42 monomer solution (Figure 3a) to assess the relationship between |∆Raman shift| and incubation time. Qualitatively similar behavior was observed for various probe surface densities in terms of temporal response, with the frequency shift reaching a maximum and saturating for affinity peptide incubation solutions > 0.1 mg/mL (Figure 3c). Figure 3b and d show the absolute change in the DSNB nitro stretch Raman shift, |∆Raman shift|, after incubation of the AgNF/DSNB/affinity peptide capture probe in solutions of Aβ42 monomer for different incubation times and different temperatures. Different aggregation dynamics were observed at different temperatures. The most pronounced frequency shift observed for the interaction of Aβ42 monomer with the P1-bound sensor occurs at room temperature (RT) after an incubation time of ~ 1 h. The shift decreases at longer times (discussed further on). The absolute value of shift change could reach maximum at 37°C at the very beginning, around 10 min, which could be due to the faster aggregation rate of Aβ42 monomer at higher temperature. While significant frequency shift was observed at longer time ( ~ 2 h) for incubation of Aβ42 with P1 at 4°C, suggesting the lower dynamic aggregation of Aβ42 at lower temperature. It results in |∆Raman shift| of 0.5 cm-1, coinciding with the case of P2 within time span of 150 min from beginning (Figure 3d, blue curve). Qualitatively similar behavior was observed for fiber sensing except a longer incubation time (~5 h) was required to reach the maximum frequency shift for the incubation at room temperature. The incubation time was expected to be shorter (~3 h) and longer (~ 8 h) when the frequency shift reaching the maximum at 37°C and 4°C, respectively, as shown in Figure 3d.


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Thus, substrate incubation with 0.1 mg/mL affinity peptides for the modification of the DSNB/AgNF substrates, and room temperature incubation of the substrates for 1 h and 5 h with Aβ42 monomer or fiber respectively was selected as the optimal experimental conditions for the Aβ42 monomer/aggregate detection.

Figure 3. Dependence of |∆ Raman shift| on incubation time of Aβ42 with peptide/DSNB/AgNF at different probe incubation concentrations (surface densities) for P1 (a) and P2 (c) sensing, respectively.

Dependence

of

|∆Raman

shift|

on

incubation

time

of

Aβ42

with


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Peptide/DSNB/AgNF at different temperatures for P1 (b) and P2 (d), respectively. The concentration of Aβ42 momomer solution used here is 4.6 × 10-6 mol/L.

The SERS spectral changes associated with Aβ42 monomer/fiber sensing in solution over time are shown in Figure 4a, b and d, e respectively. Upon exposure to Aβ42 solution with different incubation time, the peak position at 1334 cm−1 varied gradually. The striking spectra changes in the range of 1330 cm-1-1338 cm-1 are shown more clearly in Figure 4b. As mentioned the band at 1334 cm−1 is assigned to the symmetric nitro stretch of DSNB. Other bands are prominent including at 1556 cm−1 ascribed to the aromatic ring mode, and at 1064 cm−1 assigned to the inplane ring breathing mode coupled with ν (C−S). Raman spectra were shown in Figure 4, where the absolute change of Raman shift upon different incubation temperature of P1 and P2 were clearly presented after the probing substrate exposure to 4.6 h 10-6 M Aβ42 momomer solution. In contrast to our previous work51 and other reported results,52, 53 the 1334 cm−1 band was upshifted with increasing incubation time and reached a maximum |• Raman shift| value at ~1 h, then downshifted gradually. The distinct tendency is depicted in Figure 4c. We observed a similar phenomenon for Aβ42 fiber recognition by P2. For probe P2, the band at 1334 cm−1 was observed to upshift from 0 min to 5 h and downshift afterwards as shown in Figure 4d-f. The unusual time dependence of the Raman frequency shift observed is attributed to dynamic aggregation processes of Aβ42 in solution. The mechanism behind the Raman frequency shift has previously been attributed to a change in mechanical force in the Raman reporter bond due to target binding, though a change is solvation environment upon binding could also lead to the


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shift.51-53 The frequency shift behavior observed for P1 affinity peptide binding to the Aβ42 monomer could be understood as follows. Initially the probe interacts with the Aβ42 monomer and due to the relatively light mass and small size of the latter, the frequency shift is subtle. However as the bound Aβ42 aggregates with other monomers in solution, both the volume and mass of the oligomer forming in situ increase, resulting in a larger frequency shift (Figure 4c). After reaching maximum after 1 h reflecting continuous aggregation, the frequency shift begins to decrease gradually. This could be attributed to detachment of the oligomers from P1 probes at a certain mass threshold as the interaction site on Aβ42 oligomer becomes occupied by selfinteractions. For the interaction between the P2 probe substrate and Aβ42 fibers, the maximum shift value was observed when the incubation time is ~ 5 h. The longer time to reach maximum frequency shift than Aβ42 monomer is reasonable as fiber formation takes longer time in solution. The band was observed to downshift as presented in Figure 4f, which could be also attributed to mass increasing. To exclude the influence of affinity peptide on the Aβ42 aggregation during sensing, a control experiment in the absence of P2 were performed and the results were shown in Figure S1. Upon exposure P2 modified AgNFs to Aβ42 solution (previously stayed at RT for 3 h, 4 h and 5 h to allow free aggregation of Aβ42, respectively) for 1h, |∆ Raman shift| of DSNB at 1334 cm-1 was observed to be 0.71, 0.85 and 0.74 cm-1, respectively. The shift changes at specific time were in agreement with the case in the presence of P2, indicating the affinity peptides had no pronounced effect on the Aβ42 aggregation. These results suggest that aggregation processes of Aβ42 can be followed in solution by the frequency shift SERS method, a process that is vital to revealing the occurrence and development of AD but which otherwise can be hard to detect. The sensor can clearly


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differentiate Aβ42 monomers or oligomers from fibers efficiently and potentially identify its aggregates with specific mass through their binding at specific time.

Figure 4. Response of probes to Aβ42 incubation time. (a) SERS spectra of the P1/DSNB/AgNF substrate at di erent times after incubation with Aβ42. (b) Zoom-in of the SERS peak in (a) around 1334 cm−1. (c) Plot of the frequency shift in the ~1334 cm−1 peak in the SERS spectra in (b) as a function of the incubation time. (d) SERS spectra of P2/DSNB/AgNF substrate at di erent times after incubation with Aβ42. (e) Zoom-in of SERS peak in (d) around 1334 cm−1. (f) Plot of the variation in the peak position at ~1334 cm−1 in the SERS spectra in (e) as a function of the incubation time. To confirm that the dynamic SERS frequency shifts observed do indeed give insight into the AB42 aggregation process, the morphology of Aβ42 aggregation was imaged with AFM after incubation Aβ42 in Tris-HCl buffer over several hours. As shown in Figure 5, the morphology of


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native Aβ42 presents as particles of diameter ~3.0 nm. After incubation for 60 min, pronounced aggregation was observed and many more particles were formed on the mica surface with diameters around 5.9 nm, likely due to Aβ42 oligomer formation. These particles contribute to the maximum |∆ Raman shift| value observed during SERS detection with the P1 affinity peptide substrate. Larger particles with diameter of ~10.7 nm were observed at 180 min, which may consist of several misfolded Aβ42 monomers in a loosely aggregated form. After 300 min incubation, these larger particles tended to aggregate into tighter fibrillar morphologies with heights ~7.8 nm. The latter may correspond to the initial amyloid fiber of Aβ42 as reported by recent simulation54 and previous dynamic observation.55 These amyloid fibers were sensed by the P2 probe substrate and showed a peak value of |∆ Raman shift| at 300 min. Worthy of note is that many more Aβ42 fibers emerged over the next 60 minutes in AFM studies, linking together to form a network-like structure. These amyloid fiber networks formed amyloid plaques after incubation for 9 hours, and the plaques maturated after 20 hours. The time between 300 min and 360 min may be significant for the formation of toxic Aβ42 ongoing protofibrils,56 and this morphology can be captured by AFM and responded by SERS using the fiber affinity peptide P2. On the basis of both SERS and AFM results, we propose a possible mechanism of Aβ42 aggregation process in solution as shown in Scheme 1. At the beginning, Aβ42 monomers with flexible conformations were the majority component in solution and they were recognized and captured by P1 affinity peptide via π-π stacking with the relatively stable helix structure of Aβ42 monomers. With increasing incubation time, Aβ42 folded and aggregated into small oligomeric species. After ~1 h, the oligomers grew larger and larger, finally detaching from the P1 substrate. At around 5 h, the mature fibers started to form in solution which were then selectively captured by the P2 affinity peptide substrate. Further incubation facilitated the connection of fibers


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forming a network structure (AFM images in Figure 5). The relatively large network tended to drive the fibers off the substrate since the interaction was no longer strong enough to support such a large object.


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In Situ Monitoring the Aggregation Dynamics of Amyloid-Î² Protein

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