In recent years, Alzheimer’s disease (AD) is paid more and more attention for its terrible influence 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 Zn2+, Ca2+ are present along with Aβ in the senile plaques of AD 8, 9, where Al3+ 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 significance 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 (Zn2+、Ca2+、Al3+) 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 find 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-modified 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 film were studied. Next, we monitored the nanostructure changes of Aβ in the presence and absence of metallic ions (Zn2+、Ca2+、Al3+) 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.