www.ietdl.org Published in IET Nanobiotechnology Received on 12th November 2013 Revised on 26th December 2013 Accepted on 28th January 2014 doi: 10.1049/iet-nbt.2013.0071
ISSN 1751-8741
Interactions between gold nanoparticles and amyloid β25–35 peptide Jian Peng, Jian Weng, Lei Ren, Li-Ping Sun Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, People’s Republic of China E-mail: [email protected]
Abstract: Amyloid β25–35 (Aβ25–35) peptide is a peculiar peptide for its rapid aggregation properties and high neurotoxicity in Alzheimer’s disease. Here, the interactions between gold nanoparticles (GNPs) and Aβ25–35 monomers, oligomers and fibrils are explored under different molar ratio, temperature and pH by ultraviolet–visible and circular dichroism spectra, thioflavin T fluorescence assay and transmission electron microscope. It is concluded that Aβ25–35 can induce the aggregation of GNPs at certain concentration of Aβ25–35 monomer or oligomer. But at higher concentration of Aβ25–35, GNPs aggregates dissociate again. Furthermore, the aggregation rate increases at higher temperature or for lower pH. These results might provide the basis of a simple diagnostic tool for detecting Alzheimer’s disease.
1
Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder and the most common form of dementia among people over the age of 65 years. The development of AD is driven by the accumulation and deposition of β-amyloid peptide (Aβ) aggregates in the brain. The amyloid precursor peptide (APP) is degraded by several proteases, α-, β- and γ-secretases. A sustained imbalance between production and clearance of Aβ40–42 fragments by β- and γ-secretases leads to the accumulation of Aβ monomers, oligomers and finally large aggregated Aβ plaques composed of insoluble fibrillar Aβ [1]. Aβ monomer is produced naturally in normal aging humans and it is non-toxic. However, in AD patients Aβ monomer undergoes conformational changes from a random coil to a β-sheet structure to form soluble Aβ oligomers. Aβ oligomers progress to form Aβ fibrils [2]. Aβ monomers are soluble and contain short regions of beta sheet and polyproline II helix secondary structures in solution [3], although they are largely alpha helical in membranes [4]. Aβ oligomers refer to assemblies ranging from dimers to 24-mers, or even those of higher molecular weight. The size of oligomers is distributed over a wide range from 10 kDa to 100 kDa [5]. Aβ fibrils are thread-like protein aggregates that are insoluble and resistant to protease activity. Both Aβ oligomers and fibrils are highly toxic, while soluble Aβ oligomers are reportedly more cytotoxic than Aβ fibril in general [6]. Yokoyama’s group [7–9] found that Aβ1–40-coated GNPs exhibited a reversible colour change as the pH was externally altered between 4 and 10. They interpreted that the reversible process takes place when hydrophilic Aβ possesses a three-dimensional network containing both β-sheet and α-helices. Another group investigated the effect of GNPs on Aβ1–40 fibril in detail [10], and found that IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
negatively charged GNPs could serve as nano-chaperones to inhibit the fibrillisation of Aβ1–40 peptide. Furthermore, functionalised GNPs [11–13] were also used for the interaction with Aβ peptide. Li’s group [14] investigated the influence of nanoparticles with different sizes and surface functionalities on the self-assembling fibrillogenesis of Aβ1–40 peptide, and concluded that nanoparticles of various functionalities played a critical regulatory role on the fibrillation of Aβ1–40 peptide. The detection of Aβ is helpful in early diagnosis of AD. Conventional ELISA or blotting assays cannot identify trace amount of Aβ in cerebrospinal fluid or plasma. Some simple and fast colorimetric methods using gold nanoparticles (GNPs) have been reported. Wang’s group [15, 16] developed a streptavidin functionalised GNPs-based resonance light-scattering assay and a dot-blot immunoassay for Aβ1–42 detection. Although the sensitivity reached as low as 50 pg ml−1 in aqueous solution, only Aβ monomer was checked in these assays, which exists in both healthy people and AD patients. Until now, the interactions of GNPs with Aβ in different conformations have not been investigated. Since Aβ oligomers and fibrils are more important biomarker for AD diagnosis [17–23], we studied the interactions between GNPs and Aβ monomers, oligomers and fibrils in detail. Aβ25–35 is the shortest peptide sequence that retains biological activity comparable with that of full-length Aβ1–42 [24]. Moreover, this peptide is present in senile plaques and degenerating hippocampal neurons in AD brains, but not in normal control subjects [25]. Certain forms of Aβ1–40 can be converted to Aβ25–35 peptide by brain proteases [26]. Aβ25–35 contains the functional domain (sequence GSNKGAIIGLM) of the Aβ precursor protein that is required both for neurotrophic effects in normal neural tissues and also involved in the neurotoxic effects in AD [27, 28]. So, Aβ25–35 has often been chosen as a model 295
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www.ietdl.org for full-length Aβ in structural and functional studies. Therefore, we used Aβ25–35 peptide to investigate its interaction with GNPs. Multispectroscopic methods were used in the present work, including ultraviolet–visible (UV– Vis) and circular dichroism (CD) spectra, thioflavin T (ThT) fluorescence assay and transmission electron microscopy (TEM). The absorption spectrum change, morphology change and fluorescence intensity change were determined. The conformational changes of Aβ25–35 monomers, oligomers and fibrils during the binding process were investigated.
2
Experimental
Dimethyl sulfoxide (DMSO), hexafluoro-2-propanol (HFIP) and ThT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid trihydrate (HAuCl4·3H2O), NaOH (sodium hydroxide), hydrochloric acid (HCl) and trisodium citrate were purchased from Sinopharm Chemical (Shanghai, China). Aβ25–35 (Mw = 1060.29 Da) was purchased from GL Biochem Ltd (Shanghai, China). All chemicals were of analytical reagent grade, and high-purity water (18 MΩ/cm) was used throughout. GNPs were synthesised from the reduction of HAuCl4 solution by trisodium citrate as carried in our previous report [29]. Briefly, 94 ml of water and 5 ml of 1% trisodium citrate were mixed well, and 1 ml of 1% HAuCl4 was then added to the mixture. The resulting mixture was stirred and boiled in a 700-W microwave oven on high power for 1 min, and then kept heating on medium power for 5 min until the colour of the solution changed to wine red. The heated GNPs solution was kept at room temperature until cooled and then stored in the refrigerator (4°C). During the reduction, the colour of the sample was changed from faint yellowish to colourless, and then wine-red. The concentration of GNPs solution was determined using the absorbance values at 520 nm with the extinction coefficient of 2.7 × 108 M−1 cm−1 [30]. To prepare the Aβ25–35 stock, lyophilised peptide was treated with 100% HFIP to eliminate pre-existing Aβ aggregates [31]. The stock was sonicated for 10 min, and then HFIP was removed under vacuum overnight. After HFIP desiccation, a thin film of Aβ was formed. The Aβ thin film was then dissolved by 5 mM DMSO. Freshly resuspended Aβ25–35 was then diluted with cold H2O to a final concentration of 100 µM to produce Aβ monomers that should be used up within an hour [32]. Aβ oligomers were prepared by Minkeviciene’s method [33] with a slight modification. Freshly resuspended 5 mM Aβ25–35 was diluted with 10 mM cold phosphate buffer (pH = 7.4) to 100 µM, vortex for 15 s, transfer to 4°C and incubate for 24 h to produce Aβ oligomers. Dilute the freshly resuspended 5 mM Aβ25–35 with 10 mM phosphate buffer (pH = 7.4) to 100 µM final Aβ, vortex for 15 s, transfer to 37°C and incubate for 24 h to produce Aβ fibrils [34]. Aβ25–35 monomer, oligomer or fibril were mixed with GNPs, respectively, vortexed for 10 s and incubated for 30 min before UV–Vis, fluorescence, CD spectra and TEM photographs were obtained. The UV–Vis absorption spectra were measured with a Beckman DU 800 spectrophotometer (Brea, California, USA), equipped with 1 cm quartz cells. The spectra were recorded in the wavelength range of 400–800 nm, at a scan speed of 400 nm min−1. The CD spectra were measured from 190 to 260 nm at room temperature on a Jasco J-810 spectrometer (Tokyo, Japan) using a cell with a path length 296 & The Institution of Engineering and Technology 2014
of 0.1 cm. Data were collected every 0.2 nm with 3 nm bandwidth, at a scan speed of 50 nm min−1 and response time of 8 s. All spectra were collected in a triplicate and a background-corrected against pure water blank. ThT fluorescence assay: The confirmation change of Aβ25–35 fibrils was measured using ThT fluorescence assay. ThT is a classic amyloid dye that are widely used to probe Aβ fibril. Upon binding to fibrils, ThT experiences a large enhancement of its fluorescence emission [35]. ThT was prepared freshly as required in ultra-pure water as a 1 mM stock solution. Before use, the stock solution was diluted into assays at a concentration of 8 μM. 4 μM Aβ25–35 was mixed with GNPs and incubated in a 1.5 ml brown centrifuge tube with ThT. ThT fluorescence intensities were measured by a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) at 25°C. ThT emission was monitored at 485 nm with excitation at 445 nm. Three replicates were performed and the data were averaged. The standard deviations were calculated. Atomic force microscopy (AFM) assay: AFM measurements were performed using Nanoscope V multimode atomic force microscope (Veeco Instruments, USA). The samples for AFM images were diluted with deionised H2O to yield a final concentration of 1 μM. Then the sample (30 μl) was applied onto freshly cleaved muscovite mica and allowed to dry. Tapping mode was used to acquire the images under ambient conditions. Transmission electron microscopy: GNPs solution was dropped onto a 400-mesh Formvar carbon-coated copper grid (EMS Inc., Hatfield, PA, USA) and dried at room temperature. The samples were examined under the JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) with an accelerating voltage of 100 kV.
3 3.1
Results and discussion Samples characterisation
3.1.1 Characterisation of GNPs: GNPs solution exhibited a colour of wine red (Fig. 1a). The sodium citrate was served as both reducing agent and stabiliser of GNPs by yielding a negatively charged gold surface. The UV–Vis spectrum exhibited a narrow and symmetric absorption band at 520 nm (Fig. 1b). Fig. 1c showed spherical shape of GNPs with particle size of 15 ± 2.8 nm. The zeta-potential of GNPs was −38.4 ± 1.5 mV at pH 5, indicating negatively charged surface of citrate-stabilised GNPs. 3.1.2 Characterisation of Aβ25–35 monomer, oligomer and fibril: The differences between Aβ25–35 monomers, oligomers and fibrillar Aβ are evident in AFM images (Figs. 2a–c). Although the monomers cannot be clearly seen in Fig. 2a, the height is about 0.2–0.6 nm. Soluble Aβ25–35 oligomers appeared as completely fibril-free small globular structures (diameter < 6 nm) (Fig. 2b). Aβ25–35 fibrils, however, showed long thread-like structures (diameter < 12 nm, length > 1 µm) (Fig. 2c). The structures of three distinct Aβ species were further confirmed by CD spectra (Fig. 2d ). Aβ25–35 fibril showed a biphasic wave with the positive peak near 192 nm and the negative peak near 213 nm; the positive and negative peaks correspond with a typical β-turn structure and β-sheet structure, respectively. However, that of Aβ25–35 monomer showed features of random coil conformation. Compared with the monomer, the negative peak of Aβ25–35 oligomer experienced a red-shift from 198 to 205 nm, indicating the IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 1
Characterisation of GNPs
a Optical photo b UV–Vis absorption spectra c TEM image of GNPs
formation of β-sheet structure, whereas the negative peak of Aβ25–35 fibril continued to shift to 213 nm, indicating the β-sheet structures formed completely. These data confirmed that Aβ25–35 monomer, oligomer and fibril have been successfully prepared. The amyloid formation process is described in Fig. 3. The aggregation of Aβ has two kinetic
phases. In the ‘nucleation phase’, monomeric Aβ with random-coil structure undergoes conformational change and associates to form dimers, and then aggregates into oligomeric nuclei (oligomers). In the ‘elongation phase’, oligomers rapidly grow and form larger aggregates known as fibrils [36, 37].
Fig. 2 Characterisation of Aβ25–35 monomer, oligomer and fibril a AFM image of monomers b AFM image of oligomers c AFM image of fibrils d CD spectra of Aβ25–35 monomers, oligomers and fibrils IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 3 Amyloid formation process from monomers, oligomers to fibrils
3.2 Interaction of GNPs with Aβ25–35 monomers, oligomers and fibrils 3.2.1 Interaction of GNPs with Aβ25–35 monomers: UV–Vis spectra of 1 nM GNPs with different concentrations of Aβ25–35 monomer were measured. When the Aβ25–35 monomer concentration was increased to 0.6 μM, a significant peak between 600 and 650 nm appeared (Fig. 4a). The typical change of A650/A520 (Fig. 4b) has been assigned to GNPs aggregation [38]. Interestingly, when Aβ25–35 monomer concentration reached 50 μM, the peak shifted back to 520 nm. Aβ25–35 has an isoelectric point of about 10.1 and carries a net positive charge at pH 7. The GNPs are negatively charged due to the surface-adsorbed citrate anions. When the concentration of Aβ25–35 was below 0.3 μM, the system remained stable due to electrostatic repulsion between GNPs. At 0.3–10 μM, the electrostatic attraction between positively charged Aβ25–35 monomers and negatively charged GNPs would induce the
aggregation of GNPs. As Aβ25–35 monomer concentration increased to 10 μM, GNPs were totally covered with positively charged Aβ25–35. Thus the electrostatic repulsion between Aβ25–35-coated GNPs stabilised the system, resulting in dispersed nanoparticles again. TEM results further confirmed the process (Fig. 4c). CD spectroscopy was used to measure the secondary structure change of Aβ25–35 monomers after interaction with GNPs. 50 μM Aβ25–35 monomers were incubated with 0.2 nM and 1 nM GNPs, respectively. It was found that GNPs could promote the change of random-coil to β-sheet structure of Aβ, and the change is dependent on the concentration of GNPs (Fig. 5a). After incubation with 0.2 nM GNPs, the band of Aβ25–35 monomers experienced a red shift from 198 to 205 nm. The red shift continued when GNPs concentration increased to 1 nM. The β-sheet structure of Aβ25–35 monomers increased from 16.80 to 40.80% and random-coil structure decreased from 48.1% to 39.5% in the presence of 0.2 nM GNPs. When GNPs was
Fig. 4 Interaction of GNPs with Aβ25–35 monomers a Extinction spectra of GNPs Aβ25–35 monomers b Plot of the absorption ratio (A650/A520) against concentration of Aβ25–35 monomers. Data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated c TEM images of GNPs in the absence (control) or presence of 0.1 μM, 1 μM and 50 μM Aβ25–35 monomers. The incubation time was 30 min 298 & The Institution of Engineering and Technology 2014
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Fig. 5 Interaction of GNPs with Aβ25–35 oligomers a CD spectra of 50 μM Aβ25–35 monomers incubated with 0–1 nM GNPs b CD spectra of 50 μM Aβ25–35 oligomers with 0–1 nM GNPs c UV–Vis spectra of 1 nM GNPs with Aβ25–35 oligomers d Plot of the absorption ratio (A650 nm/A520 nm) against concentration of Aβ25–35 oligomers; data were calculated from c. Three replicates were collected and averaged; the standard deviations were calculated. The incubation time was 30 min
Fig. 6 Interaction of GNPs with Aβ25–35 fibrils a Extinction spectra of GNPs with Aβ25–35 fibrils b Plot of the absorption ratio (A650/A520) against concentration of Aβ25–35 fibrils. Data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated c TEM images and corresponding optical photographs (insert) of GNPs with different concentrations of Aβ25–35 fibrils. The incubation time was 30 min IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 7 Investigation of the effect of pH on GNPs a Extinction spectra of GNPs at pH 4–10 b Extinction spectra of GNPs with Aβ25–35 fibrils at pH 4–10 c TEM images of GNPs with Aβ25–35 fibrils at pH = 4, 7 and 10. [Aβ25–35] = 1 μM, [GNP] = 1 nM. The incubation time was 30 min
1 nM, the β-sheet was 39.3% and the random-coil decreased to 37.10%. 3.2.2 Interaction of GNPs with Aβ25–35 oligomers: Aβ25–35 oligomers-induced GNPs aggregation was monitored by UV–Vis spectra. When Aβ25–35 oligomer concentration increased from 0.1 to 5 μM, there was an increase in the plasmon absorption bands at 650 nm along with a concomitant decrease at 520 nm. However, when Aβ25–35 oligomer concentration reached 50 μM, the spectrum reverted back to its original state (Fig. 5c). The A650/A520 value gradually increased from 0.2 to 1.2 with increased concentrations of Aβ25–35 oligomers from 0.1 to 5 μM. At 50 μM Aβ25–35 oligomers, the A650/A520 value decreased to 0.3. These results indicated that GNPs
aggregated in a concentration-dependent manner. The Aβ25–35 oligomers were also incubated with various concentrations of GNPs. When GNPs concentration increased from 0 to 0.2 nM, the negative peak of CD spectra shifted gradually from 212 to 217 nm, indicating that the Aβ25–35 oligomers changed gradually from β-sheet to β-turn. The β-turn structure of Aβ25–35 oligomers increased from 4.10 to 15.40% and the β-sheet structure decreased from 55.0 to 35.30% in the presence of 0.2 nM GNPs. When GNPs was increased to 1 nM, the negative peak returned to 213 nm (Fig. 5b). The β-sheet increased to 48.1% and the β-turn decreased to 8.0% compared with 0.2 nM GNPs. 3.2.3 Interaction of GNPs with Aβ25–35 fibrils: The UV–Vis spectra showed obvious redshift from 520 to
Fig. 8 Results of GNPs after incubation a CD spectra of 50 μM Aβ25–35 fibrils and mixture of Aβ25–35 fibril with 0.1 nM to 1 nM GNPs b ThT fluorescence intensity at 485 nm. Three replicates were collected and averaged; the standard deviations were calculated. [ThT] = 8 μM, [Aβ25–35] = 4 μM. The incubation time was 30 min 300 & The Institution of Engineering and Technology 2014
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Fig. 9
Effect of temperature on the interaction between Aβ25–35 fibrils and GNPs
a Extinction spectra of GNPs with Aβ25–35 fibrils incubated at different temperatures b Plot of the absorption ratio (A650/A520) against temperature; data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated. The incubation time was 30 min
650 nm when Aβ25–35 fibrils concentration increased from 0.1 to 1 μM (Fig. 6). At 50 μM Aβ25–35 fibril, the peak reverted back to 528 nm. At 0.3 μM Aβ25–35 fibrils, the A650/A520 value was about 0.4 and the colour of the sample was red (Fig. 6c), while the A650/A520 value of 0.3 μM Aβ25–35 monomers was above 0.8 (Fig. 4b), indicating a lower aggregation rate of GNPs induced by Aβ25–35 fibrils than by Aβ25–35 monomers. Accordingly, with increasing concentrations of Aβ25–35 fibrils (0.1–1 μM), the colour of the samples changed from red to purple. At 50 μM Aβ25–35 fibrils, it turned back to red. The results indicated that GNPs gradually aggregated and became dispersed again when the concentration of Aβ25–35 fibrils reached 50 μM. TEM photographs further confirmed this process (Fig. 6c). To investigate the effect of pH on bare GNPs or Aβ25–35 fibrils-coated GNPs, we performed UV–Vis spectra and
TEM under different pH. The absorption spectra for bare GNPs solution show peaks with a maximum absorbance around 520 nm regardless of the pH between 4 and 10 (Fig. 7a). At pH 8–10, the absorption spectra of Aβ25–35 fibril-coated GNPs showed nearly the same peaks as GNPs. However, when pH was adjusted between 4 and 7, bimodal absorption peaks consisted of a band around 520 ± 1 nm (λ1) and a band between 600 and 700 nm (λ2) appeared (Fig. 7b). In the bimodal features, the λ1 component indicated the existence of free GNPs and λ2 was attributed to aggregated GNPs. λ2 red-shifted and gained more amplitude as pH decreased. It became more prominent than λ1 at pH < 7. Aβ25–35 has an isoelectric point of about 10.1 and carries a net positive charge at pH < 7. The lower the pH was adjusted, the more positive-charged Aβ25–35 fibril was, leading to more GNPs aggregation.
Fig. 10 Extinction spectra of GNPs with Aβ25–35 fibrils incubated at different temperatures a 28°C b 45°C c 75°C at different time d Plot of the absorption ratio (A650/A520) at different temperature against time; data were calculated from a, b and c. Three replicates were collected and averaged; the standard deviations were calculated IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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www.ietdl.org GNPs also influenced the secondary structure change of Aβ25–35 fibrils (Fig. 8a). After incubated with GNPs, there were slight decrease of β-sheet and random coil structures of Aβ25–35 fibrils and increase of β-turn and α-helix structures. Therefore, GNPs may inhibit the aggregation of Aβ25–35 fibrils, which was further confirmed by ThT fluorescence assay (Fig. 8b). The Aβ25–35 fibrils were mixed with various concentrations of the GNPs and monitored in the same incubation condition described above. During the incubation, all ThT intensity decreased. The decrease of ThT level of Aβ25–35 fibril alone should be due to fibril sedimentation [39]. A series of experiments were conducted to investigate the effect of temperature on the interaction between Aβ25–35 fibrils and GNPs. Aβ25–35 solutions were incubated at different temperature with GNPs for 30 min. A 10 nm red-shift was observed at 4°C. With increased temperature, bimodal absorption peaks consisted of a band around 520 nm (λ1) and a band between 600 and 700 nm (λ2) appeared (Fig. 9a). λ2 shifted to the red and gained more amplitude as temperature increased. When the temperature was over 28°C, λ2 became more prominent than λ1. Furthermore, the A650/A520 value increased with temperature. The new peak between 600 and 700 nm inspired us to explore the kinetic process of interaction between Aβ25–35 fibrils and GNPs. We incubated Aβ25–35 with GNPs under 28°C, 45°C and 75°C and monitored the UV–Vis spectra at different time (Fig. 10). In conclusion, the aggregation rate of GNPs (A650/A520) increased with both temperature and time (Figs. 9b and 10).
4
Conclusion
We explored the interaction between GNPs and different forms of Aβ25–35 peptide by UV–Vis spectra, CD spectra and TEM. The UV–Vis spectra and TEM results demonstrated that positively charged Aβ25–35 peptide in different forms could induce the aggregation of GNPs and red-to-blue colour change, which could be seen by naked eye. Furthermore, the aggregation rate depended on pH and temperature of the system. The CD spectra results indicated that GNPs could promote the secondary structure change of Aβ25–35 monomers from random coil to beta structure and that of Aβ25–35 fibrils from beta structure to α-helix. This study provided the information for the identification of different forms of Aβ peptide, thus playing an important role in the early diagnoses of Alzheimer’s disease. Our further work is to design specific modified GNPs to target Aβ oligomers – the biomarkers of Alzheimer’s disease – and to develop a convenient and fast diagnosis tool for real samples, such as cerebrospinal fluid of AD patients in clinic.
5
Acknowledgments
The authors thank the financial support from the National Natural Science Foundation of China (no. 81171453).
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34 Xu, S.P., Yang, Y.Y., Xue, D., et al.: ‘Cognitive-enhancing effects of polygalasaponin hydrolysate in Aβ25–35-induced amnesic mice’, Evid. Based Complement Alternat. Med., 2011, 2011, pp. 839720–839731 35 Biancalana, M., Koide, S.: ‘Molecular mechanism of Thioflavin-T binding to amyloid fibrils’, Biochim. Biophys. Acta, 2010, 1804, pp. 1405–1412 36 Outeiro, T.F.: ‘FlAsH illuminates Aβ aggregation’, Nat. Chem. Biol., 2011, 7, pp. 581–582 37 Kumar, S., Rezaei-Ghaleh, N., Terwel, D., et al.: ‘Extracellular phosphorylation of the amyloid beta-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer’s disease’, EMBO J., 2011, 30, pp. 2255–2265 38 Chen, C.E., Zhao, C.Q., Yang, X.J., Ren, J.S., Qu, X.G.: ‘Enzymatic manipulation of DNA-modified gold nanoparticles for screening G-quadruplex ligands and evaluating selectivities’, Adv. Mater., 2010, 22, pp. 389–393 39 Nilsson, M.R.: ‘Techniques to study amyloid fibril formation in vitro’, Methods, 2004, 34, pp. 151–160
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ISSN 1751-8741
Interactions between gold nanoparticles and amyloid β25–35 peptide Jian Peng, Jian Weng, Lei Ren, Li-Ping Sun Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, People’s Republic of China E-mail: [email protected]
Abstract: Amyloid β25–35 (Aβ25–35) peptide is a peculiar peptide for its rapid aggregation properties and high neurotoxicity in Alzheimer’s disease. Here, the interactions between gold nanoparticles (GNPs) and Aβ25–35 monomers, oligomers and fibrils are explored under different molar ratio, temperature and pH by ultraviolet–visible and circular dichroism spectra, thioflavin T fluorescence assay and transmission electron microscope. It is concluded that Aβ25–35 can induce the aggregation of GNPs at certain concentration of Aβ25–35 monomer or oligomer. But at higher concentration of Aβ25–35, GNPs aggregates dissociate again. Furthermore, the aggregation rate increases at higher temperature or for lower pH. These results might provide the basis of a simple diagnostic tool for detecting Alzheimer’s disease.
1
Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder and the most common form of dementia among people over the age of 65 years. The development of AD is driven by the accumulation and deposition of β-amyloid peptide (Aβ) aggregates in the brain. The amyloid precursor peptide (APP) is degraded by several proteases, α-, β- and γ-secretases. A sustained imbalance between production and clearance of Aβ40–42 fragments by β- and γ-secretases leads to the accumulation of Aβ monomers, oligomers and finally large aggregated Aβ plaques composed of insoluble fibrillar Aβ [1]. Aβ monomer is produced naturally in normal aging humans and it is non-toxic. However, in AD patients Aβ monomer undergoes conformational changes from a random coil to a β-sheet structure to form soluble Aβ oligomers. Aβ oligomers progress to form Aβ fibrils [2]. Aβ monomers are soluble and contain short regions of beta sheet and polyproline II helix secondary structures in solution [3], although they are largely alpha helical in membranes [4]. Aβ oligomers refer to assemblies ranging from dimers to 24-mers, or even those of higher molecular weight. The size of oligomers is distributed over a wide range from 10 kDa to 100 kDa [5]. Aβ fibrils are thread-like protein aggregates that are insoluble and resistant to protease activity. Both Aβ oligomers and fibrils are highly toxic, while soluble Aβ oligomers are reportedly more cytotoxic than Aβ fibril in general [6]. Yokoyama’s group [7–9] found that Aβ1–40-coated GNPs exhibited a reversible colour change as the pH was externally altered between 4 and 10. They interpreted that the reversible process takes place when hydrophilic Aβ possesses a three-dimensional network containing both β-sheet and α-helices. Another group investigated the effect of GNPs on Aβ1–40 fibril in detail [10], and found that IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
negatively charged GNPs could serve as nano-chaperones to inhibit the fibrillisation of Aβ1–40 peptide. Furthermore, functionalised GNPs [11–13] were also used for the interaction with Aβ peptide. Li’s group [14] investigated the influence of nanoparticles with different sizes and surface functionalities on the self-assembling fibrillogenesis of Aβ1–40 peptide, and concluded that nanoparticles of various functionalities played a critical regulatory role on the fibrillation of Aβ1–40 peptide. The detection of Aβ is helpful in early diagnosis of AD. Conventional ELISA or blotting assays cannot identify trace amount of Aβ in cerebrospinal fluid or plasma. Some simple and fast colorimetric methods using gold nanoparticles (GNPs) have been reported. Wang’s group [15, 16] developed a streptavidin functionalised GNPs-based resonance light-scattering assay and a dot-blot immunoassay for Aβ1–42 detection. Although the sensitivity reached as low as 50 pg ml−1 in aqueous solution, only Aβ monomer was checked in these assays, which exists in both healthy people and AD patients. Until now, the interactions of GNPs with Aβ in different conformations have not been investigated. Since Aβ oligomers and fibrils are more important biomarker for AD diagnosis [17–23], we studied the interactions between GNPs and Aβ monomers, oligomers and fibrils in detail. Aβ25–35 is the shortest peptide sequence that retains biological activity comparable with that of full-length Aβ1–42 [24]. Moreover, this peptide is present in senile plaques and degenerating hippocampal neurons in AD brains, but not in normal control subjects [25]. Certain forms of Aβ1–40 can be converted to Aβ25–35 peptide by brain proteases [26]. Aβ25–35 contains the functional domain (sequence GSNKGAIIGLM) of the Aβ precursor protein that is required both for neurotrophic effects in normal neural tissues and also involved in the neurotoxic effects in AD [27, 28]. So, Aβ25–35 has often been chosen as a model 295
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www.ietdl.org for full-length Aβ in structural and functional studies. Therefore, we used Aβ25–35 peptide to investigate its interaction with GNPs. Multispectroscopic methods were used in the present work, including ultraviolet–visible (UV– Vis) and circular dichroism (CD) spectra, thioflavin T (ThT) fluorescence assay and transmission electron microscopy (TEM). The absorption spectrum change, morphology change and fluorescence intensity change were determined. The conformational changes of Aβ25–35 monomers, oligomers and fibrils during the binding process were investigated.
2
Experimental
Dimethyl sulfoxide (DMSO), hexafluoro-2-propanol (HFIP) and ThT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid trihydrate (HAuCl4·3H2O), NaOH (sodium hydroxide), hydrochloric acid (HCl) and trisodium citrate were purchased from Sinopharm Chemical (Shanghai, China). Aβ25–35 (Mw = 1060.29 Da) was purchased from GL Biochem Ltd (Shanghai, China). All chemicals were of analytical reagent grade, and high-purity water (18 MΩ/cm) was used throughout. GNPs were synthesised from the reduction of HAuCl4 solution by trisodium citrate as carried in our previous report [29]. Briefly, 94 ml of water and 5 ml of 1% trisodium citrate were mixed well, and 1 ml of 1% HAuCl4 was then added to the mixture. The resulting mixture was stirred and boiled in a 700-W microwave oven on high power for 1 min, and then kept heating on medium power for 5 min until the colour of the solution changed to wine red. The heated GNPs solution was kept at room temperature until cooled and then stored in the refrigerator (4°C). During the reduction, the colour of the sample was changed from faint yellowish to colourless, and then wine-red. The concentration of GNPs solution was determined using the absorbance values at 520 nm with the extinction coefficient of 2.7 × 108 M−1 cm−1 [30]. To prepare the Aβ25–35 stock, lyophilised peptide was treated with 100% HFIP to eliminate pre-existing Aβ aggregates [31]. The stock was sonicated for 10 min, and then HFIP was removed under vacuum overnight. After HFIP desiccation, a thin film of Aβ was formed. The Aβ thin film was then dissolved by 5 mM DMSO. Freshly resuspended Aβ25–35 was then diluted with cold H2O to a final concentration of 100 µM to produce Aβ monomers that should be used up within an hour [32]. Aβ oligomers were prepared by Minkeviciene’s method [33] with a slight modification. Freshly resuspended 5 mM Aβ25–35 was diluted with 10 mM cold phosphate buffer (pH = 7.4) to 100 µM, vortex for 15 s, transfer to 4°C and incubate for 24 h to produce Aβ oligomers. Dilute the freshly resuspended 5 mM Aβ25–35 with 10 mM phosphate buffer (pH = 7.4) to 100 µM final Aβ, vortex for 15 s, transfer to 37°C and incubate for 24 h to produce Aβ fibrils [34]. Aβ25–35 monomer, oligomer or fibril were mixed with GNPs, respectively, vortexed for 10 s and incubated for 30 min before UV–Vis, fluorescence, CD spectra and TEM photographs were obtained. The UV–Vis absorption spectra were measured with a Beckman DU 800 spectrophotometer (Brea, California, USA), equipped with 1 cm quartz cells. The spectra were recorded in the wavelength range of 400–800 nm, at a scan speed of 400 nm min−1. The CD spectra were measured from 190 to 260 nm at room temperature on a Jasco J-810 spectrometer (Tokyo, Japan) using a cell with a path length 296 & The Institution of Engineering and Technology 2014
of 0.1 cm. Data were collected every 0.2 nm with 3 nm bandwidth, at a scan speed of 50 nm min−1 and response time of 8 s. All spectra were collected in a triplicate and a background-corrected against pure water blank. ThT fluorescence assay: The confirmation change of Aβ25–35 fibrils was measured using ThT fluorescence assay. ThT is a classic amyloid dye that are widely used to probe Aβ fibril. Upon binding to fibrils, ThT experiences a large enhancement of its fluorescence emission [35]. ThT was prepared freshly as required in ultra-pure water as a 1 mM stock solution. Before use, the stock solution was diluted into assays at a concentration of 8 μM. 4 μM Aβ25–35 was mixed with GNPs and incubated in a 1.5 ml brown centrifuge tube with ThT. ThT fluorescence intensities were measured by a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) at 25°C. ThT emission was monitored at 485 nm with excitation at 445 nm. Three replicates were performed and the data were averaged. The standard deviations were calculated. Atomic force microscopy (AFM) assay: AFM measurements were performed using Nanoscope V multimode atomic force microscope (Veeco Instruments, USA). The samples for AFM images were diluted with deionised H2O to yield a final concentration of 1 μM. Then the sample (30 μl) was applied onto freshly cleaved muscovite mica and allowed to dry. Tapping mode was used to acquire the images under ambient conditions. Transmission electron microscopy: GNPs solution was dropped onto a 400-mesh Formvar carbon-coated copper grid (EMS Inc., Hatfield, PA, USA) and dried at room temperature. The samples were examined under the JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) with an accelerating voltage of 100 kV.
3 3.1
Results and discussion Samples characterisation
3.1.1 Characterisation of GNPs: GNPs solution exhibited a colour of wine red (Fig. 1a). The sodium citrate was served as both reducing agent and stabiliser of GNPs by yielding a negatively charged gold surface. The UV–Vis spectrum exhibited a narrow and symmetric absorption band at 520 nm (Fig. 1b). Fig. 1c showed spherical shape of GNPs with particle size of 15 ± 2.8 nm. The zeta-potential of GNPs was −38.4 ± 1.5 mV at pH 5, indicating negatively charged surface of citrate-stabilised GNPs. 3.1.2 Characterisation of Aβ25–35 monomer, oligomer and fibril: The differences between Aβ25–35 monomers, oligomers and fibrillar Aβ are evident in AFM images (Figs. 2a–c). Although the monomers cannot be clearly seen in Fig. 2a, the height is about 0.2–0.6 nm. Soluble Aβ25–35 oligomers appeared as completely fibril-free small globular structures (diameter < 6 nm) (Fig. 2b). Aβ25–35 fibrils, however, showed long thread-like structures (diameter < 12 nm, length > 1 µm) (Fig. 2c). The structures of three distinct Aβ species were further confirmed by CD spectra (Fig. 2d ). Aβ25–35 fibril showed a biphasic wave with the positive peak near 192 nm and the negative peak near 213 nm; the positive and negative peaks correspond with a typical β-turn structure and β-sheet structure, respectively. However, that of Aβ25–35 monomer showed features of random coil conformation. Compared with the monomer, the negative peak of Aβ25–35 oligomer experienced a red-shift from 198 to 205 nm, indicating the IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 1
Characterisation of GNPs
a Optical photo b UV–Vis absorption spectra c TEM image of GNPs
formation of β-sheet structure, whereas the negative peak of Aβ25–35 fibril continued to shift to 213 nm, indicating the β-sheet structures formed completely. These data confirmed that Aβ25–35 monomer, oligomer and fibril have been successfully prepared. The amyloid formation process is described in Fig. 3. The aggregation of Aβ has two kinetic
phases. In the ‘nucleation phase’, monomeric Aβ with random-coil structure undergoes conformational change and associates to form dimers, and then aggregates into oligomeric nuclei (oligomers). In the ‘elongation phase’, oligomers rapidly grow and form larger aggregates known as fibrils [36, 37].
Fig. 2 Characterisation of Aβ25–35 monomer, oligomer and fibril a AFM image of monomers b AFM image of oligomers c AFM image of fibrils d CD spectra of Aβ25–35 monomers, oligomers and fibrils IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 3 Amyloid formation process from monomers, oligomers to fibrils
3.2 Interaction of GNPs with Aβ25–35 monomers, oligomers and fibrils 3.2.1 Interaction of GNPs with Aβ25–35 monomers: UV–Vis spectra of 1 nM GNPs with different concentrations of Aβ25–35 monomer were measured. When the Aβ25–35 monomer concentration was increased to 0.6 μM, a significant peak between 600 and 650 nm appeared (Fig. 4a). The typical change of A650/A520 (Fig. 4b) has been assigned to GNPs aggregation [38]. Interestingly, when Aβ25–35 monomer concentration reached 50 μM, the peak shifted back to 520 nm. Aβ25–35 has an isoelectric point of about 10.1 and carries a net positive charge at pH 7. The GNPs are negatively charged due to the surface-adsorbed citrate anions. When the concentration of Aβ25–35 was below 0.3 μM, the system remained stable due to electrostatic repulsion between GNPs. At 0.3–10 μM, the electrostatic attraction between positively charged Aβ25–35 monomers and negatively charged GNPs would induce the
aggregation of GNPs. As Aβ25–35 monomer concentration increased to 10 μM, GNPs were totally covered with positively charged Aβ25–35. Thus the electrostatic repulsion between Aβ25–35-coated GNPs stabilised the system, resulting in dispersed nanoparticles again. TEM results further confirmed the process (Fig. 4c). CD spectroscopy was used to measure the secondary structure change of Aβ25–35 monomers after interaction with GNPs. 50 μM Aβ25–35 monomers were incubated with 0.2 nM and 1 nM GNPs, respectively. It was found that GNPs could promote the change of random-coil to β-sheet structure of Aβ, and the change is dependent on the concentration of GNPs (Fig. 5a). After incubation with 0.2 nM GNPs, the band of Aβ25–35 monomers experienced a red shift from 198 to 205 nm. The red shift continued when GNPs concentration increased to 1 nM. The β-sheet structure of Aβ25–35 monomers increased from 16.80 to 40.80% and random-coil structure decreased from 48.1% to 39.5% in the presence of 0.2 nM GNPs. When GNPs was
Fig. 4 Interaction of GNPs with Aβ25–35 monomers a Extinction spectra of GNPs Aβ25–35 monomers b Plot of the absorption ratio (A650/A520) against concentration of Aβ25–35 monomers. Data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated c TEM images of GNPs in the absence (control) or presence of 0.1 μM, 1 μM and 50 μM Aβ25–35 monomers. The incubation time was 30 min 298 & The Institution of Engineering and Technology 2014
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Fig. 5 Interaction of GNPs with Aβ25–35 oligomers a CD spectra of 50 μM Aβ25–35 monomers incubated with 0–1 nM GNPs b CD spectra of 50 μM Aβ25–35 oligomers with 0–1 nM GNPs c UV–Vis spectra of 1 nM GNPs with Aβ25–35 oligomers d Plot of the absorption ratio (A650 nm/A520 nm) against concentration of Aβ25–35 oligomers; data were calculated from c. Three replicates were collected and averaged; the standard deviations were calculated. The incubation time was 30 min
Fig. 6 Interaction of GNPs with Aβ25–35 fibrils a Extinction spectra of GNPs with Aβ25–35 fibrils b Plot of the absorption ratio (A650/A520) against concentration of Aβ25–35 fibrils. Data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated c TEM images and corresponding optical photographs (insert) of GNPs with different concentrations of Aβ25–35 fibrils. The incubation time was 30 min IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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Fig. 7 Investigation of the effect of pH on GNPs a Extinction spectra of GNPs at pH 4–10 b Extinction spectra of GNPs with Aβ25–35 fibrils at pH 4–10 c TEM images of GNPs with Aβ25–35 fibrils at pH = 4, 7 and 10. [Aβ25–35] = 1 μM, [GNP] = 1 nM. The incubation time was 30 min
1 nM, the β-sheet was 39.3% and the random-coil decreased to 37.10%. 3.2.2 Interaction of GNPs with Aβ25–35 oligomers: Aβ25–35 oligomers-induced GNPs aggregation was monitored by UV–Vis spectra. When Aβ25–35 oligomer concentration increased from 0.1 to 5 μM, there was an increase in the plasmon absorption bands at 650 nm along with a concomitant decrease at 520 nm. However, when Aβ25–35 oligomer concentration reached 50 μM, the spectrum reverted back to its original state (Fig. 5c). The A650/A520 value gradually increased from 0.2 to 1.2 with increased concentrations of Aβ25–35 oligomers from 0.1 to 5 μM. At 50 μM Aβ25–35 oligomers, the A650/A520 value decreased to 0.3. These results indicated that GNPs
aggregated in a concentration-dependent manner. The Aβ25–35 oligomers were also incubated with various concentrations of GNPs. When GNPs concentration increased from 0 to 0.2 nM, the negative peak of CD spectra shifted gradually from 212 to 217 nm, indicating that the Aβ25–35 oligomers changed gradually from β-sheet to β-turn. The β-turn structure of Aβ25–35 oligomers increased from 4.10 to 15.40% and the β-sheet structure decreased from 55.0 to 35.30% in the presence of 0.2 nM GNPs. When GNPs was increased to 1 nM, the negative peak returned to 213 nm (Fig. 5b). The β-sheet increased to 48.1% and the β-turn decreased to 8.0% compared with 0.2 nM GNPs. 3.2.3 Interaction of GNPs with Aβ25–35 fibrils: The UV–Vis spectra showed obvious redshift from 520 to
Fig. 8 Results of GNPs after incubation a CD spectra of 50 μM Aβ25–35 fibrils and mixture of Aβ25–35 fibril with 0.1 nM to 1 nM GNPs b ThT fluorescence intensity at 485 nm. Three replicates were collected and averaged; the standard deviations were calculated. [ThT] = 8 μM, [Aβ25–35] = 4 μM. The incubation time was 30 min 300 & The Institution of Engineering and Technology 2014
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Fig. 9
Effect of temperature on the interaction between Aβ25–35 fibrils and GNPs
a Extinction spectra of GNPs with Aβ25–35 fibrils incubated at different temperatures b Plot of the absorption ratio (A650/A520) against temperature; data were calculated from a. Three replicates were collected and averaged; the standard deviations were calculated. The incubation time was 30 min
650 nm when Aβ25–35 fibrils concentration increased from 0.1 to 1 μM (Fig. 6). At 50 μM Aβ25–35 fibril, the peak reverted back to 528 nm. At 0.3 μM Aβ25–35 fibrils, the A650/A520 value was about 0.4 and the colour of the sample was red (Fig. 6c), while the A650/A520 value of 0.3 μM Aβ25–35 monomers was above 0.8 (Fig. 4b), indicating a lower aggregation rate of GNPs induced by Aβ25–35 fibrils than by Aβ25–35 monomers. Accordingly, with increasing concentrations of Aβ25–35 fibrils (0.1–1 μM), the colour of the samples changed from red to purple. At 50 μM Aβ25–35 fibrils, it turned back to red. The results indicated that GNPs gradually aggregated and became dispersed again when the concentration of Aβ25–35 fibrils reached 50 μM. TEM photographs further confirmed this process (Fig. 6c). To investigate the effect of pH on bare GNPs or Aβ25–35 fibrils-coated GNPs, we performed UV–Vis spectra and
TEM under different pH. The absorption spectra for bare GNPs solution show peaks with a maximum absorbance around 520 nm regardless of the pH between 4 and 10 (Fig. 7a). At pH 8–10, the absorption spectra of Aβ25–35 fibril-coated GNPs showed nearly the same peaks as GNPs. However, when pH was adjusted between 4 and 7, bimodal absorption peaks consisted of a band around 520 ± 1 nm (λ1) and a band between 600 and 700 nm (λ2) appeared (Fig. 7b). In the bimodal features, the λ1 component indicated the existence of free GNPs and λ2 was attributed to aggregated GNPs. λ2 red-shifted and gained more amplitude as pH decreased. It became more prominent than λ1 at pH < 7. Aβ25–35 has an isoelectric point of about 10.1 and carries a net positive charge at pH < 7. The lower the pH was adjusted, the more positive-charged Aβ25–35 fibril was, leading to more GNPs aggregation.
Fig. 10 Extinction spectra of GNPs with Aβ25–35 fibrils incubated at different temperatures a 28°C b 45°C c 75°C at different time d Plot of the absorption ratio (A650/A520) at different temperature against time; data were calculated from a, b and c. Three replicates were collected and averaged; the standard deviations were calculated IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 295–303 doi: 10.1049/iet-nbt.2013.0071
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www.ietdl.org GNPs also influenced the secondary structure change of Aβ25–35 fibrils (Fig. 8a). After incubated with GNPs, there were slight decrease of β-sheet and random coil structures of Aβ25–35 fibrils and increase of β-turn and α-helix structures. Therefore, GNPs may inhibit the aggregation of Aβ25–35 fibrils, which was further confirmed by ThT fluorescence assay (Fig. 8b). The Aβ25–35 fibrils were mixed with various concentrations of the GNPs and monitored in the same incubation condition described above. During the incubation, all ThT intensity decreased. The decrease of ThT level of Aβ25–35 fibril alone should be due to fibril sedimentation [39]. A series of experiments were conducted to investigate the effect of temperature on the interaction between Aβ25–35 fibrils and GNPs. Aβ25–35 solutions were incubated at different temperature with GNPs for 30 min. A 10 nm red-shift was observed at 4°C. With increased temperature, bimodal absorption peaks consisted of a band around 520 nm (λ1) and a band between 600 and 700 nm (λ2) appeared (Fig. 9a). λ2 shifted to the red and gained more amplitude as temperature increased. When the temperature was over 28°C, λ2 became more prominent than λ1. Furthermore, the A650/A520 value increased with temperature. The new peak between 600 and 700 nm inspired us to explore the kinetic process of interaction between Aβ25–35 fibrils and GNPs. We incubated Aβ25–35 with GNPs under 28°C, 45°C and 75°C and monitored the UV–Vis spectra at different time (Fig. 10). In conclusion, the aggregation rate of GNPs (A650/A520) increased with both temperature and time (Figs. 9b and 10).
4
Conclusion
We explored the interaction between GNPs and different forms of Aβ25–35 peptide by UV–Vis spectra, CD spectra and TEM. The UV–Vis spectra and TEM results demonstrated that positively charged Aβ25–35 peptide in different forms could induce the aggregation of GNPs and red-to-blue colour change, which could be seen by naked eye. Furthermore, the aggregation rate depended on pH and temperature of the system. The CD spectra results indicated that GNPs could promote the secondary structure change of Aβ25–35 monomers from random coil to beta structure and that of Aβ25–35 fibrils from beta structure to α-helix. This study provided the information for the identification of different forms of Aβ peptide, thus playing an important role in the early diagnoses of Alzheimer’s disease. Our further work is to design specific modified GNPs to target Aβ oligomers – the biomarkers of Alzheimer’s disease – and to develop a convenient and fast diagnosis tool for real samples, such as cerebrospinal fluid of AD patients in clinic.
5
Acknowledgments
The authors thank the financial support from the National Natural Science Foundation of China (no. 81171453).
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