Evaluating Effect of Metallic Ions On Aggregation Behavior of Amyloid Aβ42 By AFM Imaging and SERS

Background: Excessive aggregation of β-amyloid peptides (Aβ) is regarded as the hallmark of Alzheimer’s disease. Exploring the underlying mechanism regulating Aβ aggregation remains challenging and investigating aggregation events of Aβ in the presence and absence of metal ions at molecular level would be meaningful in elucidating the role of metal cations on interactions between Aβ molecules. In this study, chemical self-assembled monolayer (SAM) method was employed to fabricate monolayer of β-amyloid peptides Aβ42 on gold substrate with a bolaamphiphile named 16-Mercaptohexadecanoic acid (MHA). Firstly, the samples of gold substrate (blank control), the MHA-modied substrate and the Aβ42-modied substrate were detected by X-ray photoelectron spectroscopy (XPS) to track the self-assembly process. Aggregation behaviors of Aβ42 before and after metallic ions (Zn 2+ (cid:0) Ca 2+ (cid:0) Al 3+ ) treated were monitored by atomic force microscopy (AFM) and the interaction between Aβ42 and metallic ions (Zn 2+ (cid:0) Ca 2+ (cid:0) Al 3+ ) was investigated by surface-enhanced Raman Scattering (SERS), respectively. Results: The XPS spectra of binding energy of gold substrate (blank control), the MHA-modied substrate and the Aβ42-modied substrate are well tted with the corresponding monolayer’s composition, which indicates that Aβ42 monolayer is well formed. The recorded surface morphology of different experimental groups obtained by AFM showed markedly different nanostructures, indicating occurrence of aggregation events between Aβ42 molecules after adding metal ions to the solution. Compared to the control group, the presence of metal ions resulted in the increased size of surface structures on the observed 3D topography. Further study by SERS showed that the Raman strength of Aβ42 changes signicantly after the metal cation treatment. A considerable part of the amide bonds interacts with metal cations, leading to a structural change, which is characterized by the weakened β-fold Raman peak. Conclusion: The AFM imaging results suggest that aggregation events occurred between Aβ42 molecules with the addition of metal cations.


Background
In recent years, Alzheimer's disease (AD) is paid more and more attention for its terrible in uence on the health of the elderly. Amyloid plaques with high density of β-amyloid peptides (Aβ) in the brain tissue of patients have been reported to be the major pathological feature of AD 1 . It was reported that the concentration of deposited Aβ in the cerebral cortex correlates with the degree of dementia and synaptic loss 2 . Main components of the plaques were studied to be Aβ40 and Aβ42, which are two homologous isomers of Aβ 3 . While Aβ40 is the most abundant homologous isomers ever discovered, Aβ42 has been reported to be the most toxic 4,5 . Hence, the article focuses on the aggregation of Aβ42, whose neurotoxicity has essential correlation with the pathology of AD 6 . There remains a key question in the pathology of AD: what are the risk factors that affect the aggregation of Aβ? In recent years, several neurotoxic metal ions were proposed as affecting factors in the misfolding and aggregation processes of Aβ 7 . It has been found through the autopsy of AD patients that abnormally high concentrations of Zn 2+ , Ca 2+ are present along with Aβ in the senile plaques of AD 8, 9 , where Al 3+ is also detected 10 .
Exploring the underlying mechanism regulating Aβ aggregation remains challenging and the mechanism of metal cations' effect on Aβ remains elusive. Until now, the lack of study on revealing molecular events of Aβ raised a controversy about whether metal cations are helpful to the aggregation of Aβ molecules. Therefore, a study on aggregation events of Aβ in the presence and absence of metal ions at molecular level would be of great signi cance in terms of pathology and methodology. In recent years, atomic force microscopy (AFM) has been widely used for studying the interaction between molecules, especially protein-protein interaction 11,12 . Since the turn of the century, new perspectives have been opened with the advent of AFM in the investigation of biomolecular interactions 13 . Up to now, some research methods have been developed to study protein-protein interactions, such as surface plasmon resonance (SPR) 14 , enzyme immunoassay (EIA) 15 , surface force apparatus (SFA) 16 , and atomic force microscopy (AFM) 17 .
Compared with other methods, AFM has the advantage of obtaining images with high spatial resolution and carrying out measurements under near physiological conditions 18 . Consequently, AFM opens novel avenues for studying pathways of interactions between proteins and makes the study of physical behaviors of proteins achievable. In addition, along with the advantage of obtaining protein molecules' topographies on nanoscale, AFM enables researchers to monitor the aggregation behaviors between Aβ monomers. This study focuses on the effects of several metal ions (Zn 2+ Ca 2+ Al 3+ ) on Aβ42 aggregation. The three-dimensional morphology measured by AFM has high spatial resolution. All experiments were carried out under near-physiological conditions and a low ion concentration, closer to physiologically relevant values, is applied. By using AFM, it's achievable to concentrate on what we can nd by "looking" at the protein molecules and analyze biological phenomena 19 .
However, the main challenge with AFM testing is the sample preparation, especially the stabilization of protein molecules onto the gold substrate. In this study, the possibility of investigating molecular events in physiological solution is realized through a sample preparation method called Self-assembly monolayer (SAM), which has been developed over the last two decades and widely applied by biologists and chemists 20,21 . To achieve SAM chemically, a cleaned gold substrate should be immersed in a solution of thiols (16-Mercaptohexadecanoic acid (MHA)), which is followed by spontaneous reaction between the thiol and gold. Gradually, MHA monolayer on the gold surface with ordered and stable bonds (Au-S bond) is formed. Afterwards, the carboxyl terminus of MHA is activated by 1-ethyl-3-(dimethylaminopropyl) carbodi-imide hydrochloride (EDC) and N-Hydroxysulfosuccinimide (NHS) and then immersed into protein (Aβ42) solution. Eventually, a stable and ordered monolayer of Aβ42 on the thiols-modi ed gold surface is formed. The mechanism of mercaptan self-assembly method is depicted in Figure 1.
In order to track the formation of Aβ monolayer, X-ray photoelectron spectroscopy (XPS) were employed.
The composition characteristics of the gold surface, the Aβ42 monolayer, and the MHA lm were studied.
Next, we monitored the nanostructure changes of Aβ in the presence and absence of metallic ions (Zn 2+ Ca 2+ Al 3+ ) by employing AFM imaging. The interaction between metallic ions and Aβ is furtherly probed and discussed by Surface-Enhanced Raman Scattering (SERS), which is an emerging sample surface analysis technique 22 . The changes of chemical bonds and groups in molecules lead to different molecular rotation or vibration states, which can be judged by the change of Raman scattering light frequency 23 . For proteins, Raman scattering can obtain not only important information about amino acid composition, but also secondary structure information such as β -sheet and α-helix. It has been reported that the Raman cross sections of Au are enhanced to some extent when they adsorb different molecules 24 . Because this effect occurs in the metal adsorbed molecular system, many important processes such as surface studies are related to it. The purpose of SERS study is to quantitatively characterize the effect of metal ions on the conformational transition of Aβ42, and to probe the role of these metal ions in the process of abnormal aggregation of Aβ42. Combined with the results of AFM study, the aggregation behavior of Aβ in the absence and presence of metallic ions was elucidated.

Results of SERS imaging
In general, the enhancement of the surface signal is 106 times, which is equivalent to the ampli cation of the surface monolayer to more than one million layers. Therefore, the advantage of SERS is that it can avoid the signal interference caused by the same substance in the solution, and obtain high-quality surface molecular vibration and rotation signals, which is of great signi cance for a detailed understanding of the interaction mode between molecules (such as metal ions) and self-assembled monolayers and the structural changes of molecules. Since the discovery of SERS, it has been successfully applied in many elds such as chemistry, biology and so on.
Above all, stable SERS signal is of great signi cance for accurate analysis results. Hence,the SERS properties of the obtained Aβ42 modi ed-substrates were evaluated in different metal ion environments.
In order to investigate the stability of Raman measurement results, the changes of SERS spectra of Aβ42 molecular layer were recorded under continuous laser irradiation. As shown in Figure 4, when different integral time was set, no obvious change in the peak shape of Raman spectrum curves for different samples was seen, which means the Aβ42 monolayer in these three metal ion solutions has good stability under continuous laser irradiation.
In Figure 5, curve A stands for the Raman spectrum of the blank control group and curve B stands for the experimental group. The results indicated that β-folds (peak at 1669cm-1) and Amide II (peak at 1375cm-1) is the characteristic structure for the natural conformation of Aβ42. With the addition of Zn 2+ , the peak intensity of the β-folded conformation at 1669cm-1 and amide II band at 1375cm-1 was weakened to a certain extent, respectively ( Figure 5(1)). Moreover, in blank control group, the Raman peak signal at 1375cm-1 was detenmined to be stronger than that at 1669cm-1, which means the vibration attributed to N-H is greater than that attributed to β-fold. On the contrary, the signal intensity of Amide II (1375cm-1) was greatly weakened after adding Zn 2+ , which is lower than that of β-fold (1669cm-1). As shown in Figure 5(2), the addition of Ca 2 + also caused a structural change of Aβ42, which is chracterized by the reduced Raman intensity of Amide II from 156.13 to 115.36, and the reduced Raman intensity of β-fold decreased from 144.669 to 117.76. It's worth noting that the Raman peak of Amide II at 1375cm-1 was greatly weakened and almost disappear, and the intensity of the β-fold peak at 1669cm-1 was also decreased compared with the blank control group, indicating that a considerable part of amide bonds in Aβ42 molecule probably interacted with Al 3+ which resulted in a conformational change ( Figure 5(3)).

Preparation of chemically immobilized Aβ42 monolayers
It's of crucial importance to consider immobilization carefully for the sensitivity and reproducibility of bioassays. Self-assembly monolayer (SAM), which is reliable for protein immobilization, is a topic of current interest in biological studies. SAM method can be used for self-assembly of protein molecules without altering the stability and activity of protein 25 . It has been studied that thiol concentration of 1 mM and immersion time of 24h are be tting for the formation of Mercaptan molecular lm 26 . Besides, MHA with a proper length of the chain serves as a spacer to minimize the interference from gold substrate 27 .
We monitored the self-assembled processes by X-ray photoelectron spectroscopy (XPS) to ensure that Aβ42 was successfully modi ed on the surface of the gold substrate. Electron emission can be observed when the sample is exposed to electromagnetic waves with short enough wavelengths, i.e. high photon energy. This phenomenon is called photoelectric effect or photoionization because of the presence of observable photocurrents. In this process, the binding energy of material can be expressed by the following equation:  Au, which reduces the binding energy of S2p 29 . The peak at 161eV may be attributed to the C-S bond 30 . The results suggest that the immobilization of Aβ42 on the gold substrate is successful.

Topographic images of Aβ42 monolayer imaged by AFM in solution
The sizes of the nanostructures on the surface of each sample were characterized by average diameter by using software analysis. The average size of the observed nanostructure in control group was calculated to be 43.12nm at the incubation time of 24h ( Figure 6 By applying software analysis, it was found that the surface roughness of Aβ42 monolayer in different experimental groups varied signi cantly. The surface roughness of each topography is characterized by an average roughness (R n ) (shown in table 1). The change of surface particles can be indicated by R n .
The results showed that the roughness of control group (in physiological solution) was 1.958 at the incubation time of 24h and little change of R n was observed at the incubation time of 48h. For Aβ42 monolayer incubated in 10 µM Zn 2+ ,Ca 2+ and Al 3+ solutions for 24 hours, the R n was calculated to be 1.762, 1.82 and 1.672, respectively. For Aβ42 monolayer incubated in 10 µM in 10 µM Zn 2+ Ca 2+ and Al 3+ solutions for 48 hours, the R n was calculated to be 1.54, 1.622 and 1.412, respectively. It can be found that with the addition of metallic ions, the decrease of R n occurred and with the increase of incubation time, R n of Aβ42 monolayer in the presence of metallic ions also decreased in varying degrees. We believe that the aggregation of Aβ42 particles leads to the collapse of molecular morphology, and more and more intermolecular gaps are covered, eventually resulting in the decrease of R n .

SERS analysis
Furthermore, the change of molecular conformation was evaluated by the Raman intensity ratio (I 1375 / I 1669 ) (displayed in Figure 7). In comparison, I 1375 / I 1669 of Aβ42 monolayer in the presence of Al 3+ is the lowest, which indicates that Al 3+ make the greatest effect on the conformation change of Aβ42. The result is consistent with Banks's study, found that Al 3+ can stabilize the aggregation structure of Aβ to a greater extent 32 .
Combined with Figure 3, the nanostructures with aggregation state on the surface in the presence of metallic ions is consistent the results obtained by SERS. Based on these results, it can be inferred the presence of metallic ions plays a vital role in the occurrence of the abnormal aggregation events of Aβ42. The implications of this phenomenon are as follows: misfolding of Aβ conformation at the early stage leads to a destabilization and the interaction between metal cations and Aβ results in a conformational change of Aβ, which promotes formation of aggregates. I t is rational to assume that to block the effects from metal cations or obstruct the interaction between Aβ and metal cations may have great potential in new drug design for AD.

Materials
Metal salts used in this work are chlorides (Zncl 2 , Cacl 2 and Alcl 3 ·6H 2 O), manufactured by Sigma Aldrich Chemical Co.. All chemicals were used as received. Phosphate buffered saline (PBS, pH 7.4) and absolute ethyl alcohol (guaranteed grade) were produced by Merck Co.. Ultra-pure water was made by Millipore

Preperation of gold lm
Besides, vapor deposition method was applied to obtain the lm of gold particles. Gold particle spray lm deposited on the mica plate in high vacuum by applying turbo evaporator (at ~10 −7 Torr), Using a radiator heater, mica plates were heated to 325℃ for 2 h prior to deposition. The velocity of evaporation is limited within the range of 0.1-0.3 nm/s. The thickness of gold granular lm is about 200 nm. In addition, a chromium lm was deposited between the gold and the surface of mica to increases the adherence.
Finally, the obtained gold lm was annealed in H 2 ame for one minute.
Before use, the prepared gold plaque was immersed in piranha solution (v/v H 2 SO 4 :H 2 O 2 =3:1) for 30 min to remove organic pollutants on the surface. Then the gold surface was washed three times by absolute ethyl alcohol and ultrapure water in turn. Next, it was dried in nitrogen to avoid any pollution.

Aβ42 immobilization onto the MHA lm
The carboxyl groups at the end of the MHA lm reacts with the amino groups of lysine in Aβ molecule 33,34 . Taking advantage of this principle, steady Aβ42 monolayer can be acquired. The Aβ protein was dissolved in a physiological solution (PBS), which was freshly prepared according to standard method. It should be noted that MHA lm were activated for 1 h at normal temperature using the method mentioned above. Afterwards, the activated MHA lm was rinsed as mentioned above and then immersed in 10 µM Aβ42 solution and stored in refrigerator at 4℃ for 12 h. At last, the Aβ42 lm was prepared. Besides, it should be pointed out that the Aβ42-modi ed substrates should be rinsed three times by ultrapure water and dried in nitrogen before testing. This step is to remove free protein molecules from the surface of the substrate. The surface of Aβ42 monolayer was characterized by XPS.

AFM imaging
The powder of the three metal salts was dissolved in PBS respectively and nally diluted to 10 µM before use. For the comparison of Aβ42 aggregation events in the absence and presence of metal cations, the Aβ42 monolayers were incubated in blank solution (PBS) and metal ionic solutions and placed in 37℃ incubator for 24h and 48h respectively. Three-dimensional images of the incubated Aβ42 monolayers were achieved at a resolution of 512×512 and a rate of 1 Hz. Surface topography of all samples were scanned by atomic force microscopy (JPK Nanowizard @ II, Germany). Three-dimensional topography of blank group and experimental groups recorded in different conditions were analyzed by imaging processing software which is offered by the company.

SERS determination
Aβ42 monolayer was prepared by the method mentioned above. 10µM concentration of metal salts was dissolved in PBS solution. The prepared Aβ42 monolayer modi ed substrate was placed in a clean liquid pool, 2 mL of blank solution (PBS) and metal ion solution (Zn 2+ Ca 2+ Al 3+ ) was added respectively, and immersed in an incubator (37℃) for 24 h.
A confocal Raman spectrometer of Horiba company was used to measure the Raman spectrum of Aβ42 molecular lm. The laser was a He Ne laser with excitation wavelength of 633nm, laser power of 6.0mw, resolution of 1cm −1 , and the measurement range was 800 ~ 2800cm −1 . With PBS solution as blank control, the conformational changes of Aβ42 with the addition of metal cations were determined by Raman spectroscopy. For each experimental group, the Raman experiment was repeated at least three times.
In addition, we used Levenbery-Marquardt algorithm for peak split and tting of the Raman spectral bands (corresponding to the β-folded conformation) (about 1600 ~ 1700cm −1 ) and the characteristic peaks corresponding to amide bond at about 1400cm −1 . During the SERS testing, the same sample parameters were set. In order to get accurate results, each sample was repeated at least three times. The integration time of Raman data was initially set as 10s, and then the integration time was gradually increased to 60s. Availability of data and material The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.    Intensity ratio I1375/I1669 of Aβ42 molecule in the absence and presence of metal cations (integration time for 10s)