3.1. 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 [28]. It has been studied that thiol concentration of 1 mM and immersion time of 24h are befitting for the formation of Mercaptan molecular film [29]. Besides, MHA with a proper length of the chain serves as a spacer to minimize the interference from gold substrate [30]. Our previous research showed that the chemical SAMs method has good reproducibility [31, 32].
We monitored the self-assembled processes by X-ray photoelectron spectroscopy (XPS) to ensure that Aβ42 was successfully modified 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:
Ek -the kinetic energy of photoelectrons (in eV); hv- the energy of photons in the X-ray source (in eV); Eb-the binding energy in the specific orbit of an atom (in eV); ϕs-the work function of the spectrometer (in eV).
The surfaces of different samples (blank substrate, MHA films, Aβ42 monolayer) were investigated by XPS and all measurements should be repeated 3 times for each sample. Full spectrum of elements obtained by XPS showed changed element binding energy of the bare gold、MHA-modified and the Aβ42-modified gold sample. The full XPS spectra of the three surfaces are shown in Figure 2. It shows that the elements on the surface of the three samples are identical to the modified molecules during self-assembly. The peak values of Au4f binding in the XPS spectra of blank substrate are 86.7 eV and 83.9 eV, respectively, which are consistent with the standard spectra. The peak values of Au4f binding shifted to 87.9eV and 90.8eV after the MHA molecule was modified on the surface of gold film. The formation of Au-S bond should be the cause of the binding-energy shift of Au4f. What’s more, peak position of the S2p binding energy spectrum (Figure 2(d)) of the MHA film is lower than 164eV, indicating that there is no unbounded MHA molecule on the sample surface [33]. There are two main peak in the binding energy spectrum of S2p. The peak at 161.2 eV may be due to the bond between MHA and Au, which reduces the binding energy of S2p [34]. The peak at 161eV may be attributed to the C-S bond [35]. The results suggest that the immobilization of Aβ42 on the gold substrate is successful.
3.2. Topographic images of Aβ42 monolayer imaged by AFM in solution
Firstly, the topography of Aβ42 monolayer in the absence of metal cations display Granular structure after incubating for 24h (Figure 3(A1)). However, the topography of Aβ42 monolayer in the presence of Zn2+, Ca2+ and Al3+ showed spherical structures without rules after 24h (Figure 3(B1)、(C1)、(D1)). On the basis of the above results, it can be suggested that in the presence of metallic ions, the aggregation behavior of Aβ42 was greatly induced; in other words, the presence of metal ions leads to disorder and clumping on the surface of Aβ42 monolayer. What’s more, stable aggregation state was observed during the experiment with the extending of incubation time. The results indicated that the addition of Zn2+, Ca2+ and Al3+ drastically destabilized Aβ42 and stabilize aggregation of Aβ42. The result is consistent with the mode proposed in Bruno’ paper [36]. Besides, the aggregation state of Aβ42 in 10 µM Ca2+、Zn2+ and Al3+solution differed in size and morphology of nanoparticles imaged by AFM. In conclusion, AFM imaging results showed large amounts of heterogeneous and conglobate-shaped aggregates were produced in the presence of metallic ions. The influence of different kinds of metal ions on the process of Aβ42 shows some differences, which may be attributed to the following reasons: 1) different charge amount, different molar ratio of Aβ42 to metal ions; 2) action modes and action sites on the Aβ42 peptide chain are different in the presence of different metal cations.
3.3. SERS analysis
In general, the enhancement of the surface signal is 106 times, which is equivalent to the amplification 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 significance for a detailed understanding of the interaction mode between molecules (such as metal ions) and self-assembled monolayers and the structural changes of molecules Righteousness. Since the discovery of SERS, it has been successfully applied in many fields such as chemistry, biology and so on.
Above all, stable SERS signal is of great significance for accurate analysis results. Hence,the SERS properties of the obtained Aβ42 modified-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 Zn2 +, 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 Zn2+, which is lower than that of β-fold (1669cm-1). As shown in Figure 5(2), the addition of Ca2 + 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 Al3+ which resulted in a conformational change.
Furthemore, the change of molecular conformation was evaluated by the Raman intensity ratio (I1375 / I1669) (displayed in Figure 6). In comparison, I1375 / I1669 of Aβ42 monoleyer in the presence of is the lowest, which indicates that Al3+ make the greatest effect on the conformation change of Aβ42. The result is consistent with Banks’s study, found that Al3+ can stabilize the aggregation structure of Aβ to a greater extent [37].
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. If we can block the abnormal aggregation of Aβ42 by chelating metal ions, we may interrupt this pathological change in the early stage of AD disease, so as to achieve the purpose of prevention and treatment of AD.