Materials Science and Engineering C 39 (2014) 227–234
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Investigation of the inhibitory effects of TiO2 on the β-amyloid peptide aggregation Mukhtar H. Ahmed a,b,⁎, John A. Byrne b, Tia E. Keyes a a b
School of Chemical Science, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast, UK
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 12 January 2014 Accepted 1 March 2014 Available online 12 March 2014 Keyword: β-Amyloid peptide TiO2 XPS Raman spectroscopy Photocatalytic
a b s t r a c t TiO2 thin films are of great interest as biocompatible coatings and also as photocatalytic self-cleaning and antimicrobial coatings. In this work we used β-amyloid as a model for infectious protein to investigate the attachment and photocatalytic degradation. TiO2 films were prepared on stainless steel substrates using magnetron sputtering. The films were characterised before and after exposure to β-amyloid (1–42), using XRD, Raman spectroscopy, XPS and AFM. The TiO2 film was mostly composed of the anatase phase with a relatively high surface roughness. The presence of Raman peaks at 1668 cm−1 and 1263 cm−1, with the XPS spectral feature for nitrogen at 400 eV, confirmed the adsorption of amyloid on surface. Following exposure of the β-amyloid contaminated TiO2 to UV-B irradiation a slight shift of amide modes was observed. Furthermore, the amide I spectra show an overall decrease in α-helix content with presence of a minor peak around 1591 cm−1, which is related to tryptophanyl and tyrosinyl radicals, which can lead to conformational change of β-amyloid. The C1s band at 292.2 eV suggests the formation of free carboxylic acid. The loss in the crucial structure of β-amyloid leads to reduce the fibril formation, thought to be induced through a photocatalytic process. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Prion diseases, known as transmissible spongiform encephalopathies (TSEs), occur in both humans (Creutzfeldt–Jakob disease (CJD)) and animals (e.g. bovine spongiform encephalopathy (BSE)). Prion diseases are a group of rapidly progressive disorders characterised by a defined spectrum of clinical abnormalities. The infectious agent consists merely of protein and is capable of replicating and transmitting infection without the need for informational nucleic acids. There has been interest in proteins causing neurodegeneration since they may all act as prions (such as β-amyloid and α-synuclein) [1]. The prion-causing agent is highly robust and has been shown to remain infectious even after modern cleaning and inactivation regimes such as autoclaving (121 °C, 30 min) or chemical methods, e.g. formaldehyde gas, have been implemented [2]. β-Amyloid (Aβ) with ~4 kDa is the major component of the amyloid plaques found in prion diseases [3]. β-Amyloid is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions (1.0 nM) [4]. The β-amyloid peptides can aggregate into soluble oligomers or fibrillar assembles with the tinctoral properties of amyloids [5]. The β-amyloid structure is extremely stable and accumulates in infected tissue (nerve cells), causing tissue damage and cell death. This ⁎ Corresponding author at: Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast, UK. E-mail address: [email protected] (M.H. Ahmed).
http://dx.doi.org/10.1016/j.msec.2014.03.011 0928-4931/© 2014 Elsevier B.V. All rights reserved.
stability means that prions are resistant to denaturation by chemical and physical agents, difficult to remove and or degrade, making disposal and containment of these proteins difficult [6]. Therefore, these diseases can be transmitted, as adverse effects of medical treatment (iatrogenic transfer) and have occurred when contaminated surgical instruments have been reused [7]. Furthermore, many medical devices cannot be effectively sterilized with classical sterilization methods due to damage by heat or chemical disinfectants [8], and such techniques are often ineffective in decontamination of potentially infectious proteins [9]. Therefore, alternative methods of surface decontamination are required. Titanium and titanium alloys are the most widely used in medical applications, because of their excellent properties including chemical stability, low density, high strength, corrosion resistance, and biocompatibility [10–12]. In-vitro and in-vivo experiments have demonstrated that titanium dioxide (TiO2) coatings exhibit good blood compatibility, and do not appear to induce platelet aggregation, damage red blood cells or adsorb fibrin strongly [13,14]. TiO2 under UV excitation exhibits photocatalytic activity wherein UV excitation promotes electrons to the conduction band creating holes in the valence band. These charge carriers can migrate to the surface and take part in redox reactions with water and molecular oxygen leading to the formation of reactive oxygen species including hydroxyl radical and superoxide radical anion. TiO2 photocatalysis has been reported to be effective for the inactivation of a wide range of pathogenic microorganisms implicated in Healthcare Associated Infections [15].
228
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
The photocatalytic degradation of proteins involves several chemical and photocatalytic stages and a great number of intermediates. Previous studies have proposed mechanisms of oxidative cleavage mediated by hydroxyl radical [OH•], which are able to attack the main and the side chains of proteins, producing molecules with lower molecular weight and finally CO2 [16]. In this work, stainless steel was coated with thin films of TiO2, followed by contamination of the surface with β-amyloid polypeptide. The photocatalytic denaturation of the β-amyloid under ultraviolet irradiation has been explored. Both adsorption and decontamination are examined using advanced surface analytical techniques, including Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). 2. Materials and methods 2.1. Preparation of TiO2 thin film Polished coupons of 316 stainless steel [48 samples of 2 × 2 cm2] were ultrasonically cleaned with acetone for 15 min, then washed using distilled water and dried with nitrogen gas. TiO2 thin film was then deposited on the coupons by radio-frequency (rf) magnetron sputtering using an unbalanced magnetron system (TEER UDP 450). Sputtering was carried out in an argon and oxygen plasma, with a Ti target (Teer, 99.6% pure) and pressure b5 × 10−6 Pa. The deposition process was carried out in the power regulation mode at 400 W, resulting in 0.98 A applied to the target. These conditions were optimised to give a maximum deposition rate. The partial pressures of reactive gas (oxygen) and working gas (argon) were controlled by the oxygen and argon flow rates. To deposit a stoichiometric film, a ratio of 15.0 cm3/min argon to 5.0 cm3/min oxygen giving 25% oxygen, was used. The deposition time was 8 h. To improve the crystallinity of the TiO2 film and obtain the anatase phase, samples were annealed at elevated temperatures in air (samples were heated at a rate of 5 °C/min to a temperature of 430 °C, held for 3 h, and cooled at 5 °C/min).
7 accumulations. This process was repeated at five different spots across the samples of TiO2 to assess the uniformity of the film. The film thickness was determined using Stylus profilometry (Dektak 8 Advanced Stylus profiler Veeco Instruments Inc., USA), automatic levelling was selected and system-based software obtained average step height calculation. Atomic force microscopy (AFM) was performed using the Digital Instruments, Veeco BioScope II. Imaging was performed under ambient conditions at 25 °C using a sharp silicon probe (R = 20 nm), attached to a low stress soft silicon nitride (SiN) cantilever with no reflecting coating on its back side [spring constant ~0.033 N/m and resonant frequency around 15 kHz and applied force of 500 mV]. The AFM exhibited a maximum scan area of 10 × 10 μm2 and a vertical range of 3 μm and was calibrated using calibration gratings purchased from Micro Masch. XPS analysis was carried out using an AXIS Ultra XPS system using a monochromatic Al-Kα X-ray source (hv 1486 eV) generated from an aluminium anode operating at an emission voltage of 15 kV with current of 5 mA at the source. The base pressure within the spectrometer during examination was 1.0 × 10−10 bar. 3. Results and discussion 3.1. TiO2 film thickness After annealing at 430 °C for 3 h, the film thickness of TiO2 samples has been determined and the values were around 100 ± 5 nm, yielding a film deposition rate of 12 nm/h. 3.2. X-ray diffraction (XRD) of TiO2 Fig. 1 shows the X-ray diffraction (XRD) analysis of TiO2 thin film, after the annealing process. The major peaks were detected at 2θ values of 25.3°, 37.9°, 48.2°, 53.0°, 55.9° and 62.6° which are corresponding to (101), (004), (200), (105), (211) and (204) anatase reflections of TiO2 respectively. The results are comparable to those obtained by Kumar et al. [17].
2.2. β-Amyloid contamination 3.3. Raman spectroscopy of TiO2
10
20
30
40
50
TiO (204) 2
TiO (105) 2
TiO (211) 2
A Bruker D8 X-ray diffractometer was used for XRD analysis. Diffraction patterns were collected in reflection-mode geometry from 20° to 80° 2θ at a rate of 0.05° 2θ/min. Raman spectra were recorded on a Horiba Jobin Yvon HR800UV microscope using ~2 mW from a 532 nm laser diode for excitation and the following parameters; confocal aperture 200 μm and spectral resolution ~ 5 cm− 1. A 100 × objective was employed and typical acquisition times were 5 s and that was repeated
TiO (200) 2
2.4. Film characterisation
TiO (004) 2
Following exposure to β-amyloid, samples were irradiated using UV-B, to determine the effect of UV photolysis and/or photocatalysis on the β-amyloid structure. The UV-B source was a fluorescent lamp (Philips, PLS 9W/12/2p) set ~ 6.0 cm above a Petri dish with peak emission at 315 nm. Dark and light control experiments were also undertaken. Dark control, samples were prepared and incubated under the same conditions but not exposed to light, by covering them with foil. Five samples were used for each time point within groups.
TiO (101) 2
2.3. UV-photocatalysis
The Raman spectrum of the TiO2 film after the annealing at 430 °C is shown in Fig. 2. The Raman active Ti\O stretching band is centred at 632 cm− 1 (Eg) and 518 cm− 1 (A1g). The bands at 400 cm− 1 and 155 cm−1, corresponded to both (B1g) and (Eg) bending vibrations of (O\Ti\O), respectively. The obtained data are consistent with anatase mode of TiO2. There was no evidence for the presence of rutile TiO2 which is characterised by modes at 606, 434 and 230 cm− 1. The Raman data correlates well with the XRD data, indicating that the TiO2
Intensity (a/u)
β-Amyloid (1–42) was obtained from Sigma Aldrich, and it was dissolved in 10 mM phosphate buffer at pH 7.4 to make a final concentration of ~ 1 × 10− 5 M and stored at − 30 °C. The samples were incubated with 50 μg/ml [~10 μM] of β-amyloid solution for 60 min at 37 ± 1 °C. At the end of the adsorption period, the samples were rinsed and washed using distilled water, and then dried.
60
2θ (degree) Fig. 1. X-ray diffraction (XRD) spectra of TiO2 thin film.
70
80
200
3.5. Atomic force microscopy of TiO2 thin film
400
600
800
The surface roughness of the TiO2 sample was characterised using AFM. Fig. 4 demonstrates the three dimensional morphology of TiO2. The annealed TiO2 film had a higher roughness than non-annealed film. The root mean square (Rq) roughness value of the deposited TiO2 film was around 23.2 nm ± 2.7. This finding is in agreement with the results published by Hazan et al. [20].
1000
Raman shift (cm-1)
3.6. Adsorption of β-amyloid onto surface of TiO2
Fig. 2. Raman spectra of TiO2 thin film.
3.6.1. Raman spectroscopy of β-amyloid Fig. 5 shows the Raman spectra of the β-amyloid adsorbed on the surface of TiO2, with and without exposure to UV-B irradiation. In the case of free β-amyloid (non-adsorbed), the vibration characteristic of the amide I band occurs at 1668 cm− 1, which involves mainly C_O stretching, C\N stretching, Cα\C\N bending, and N\H in-plane bending of α-helical peptide groups. Moreover, the amide III mode is a NH and C_O in plane bending modes with C\N stretch, and it is observed at 1263 cm− 1 [21]. The frequencies and shapes of these features are highly sensitive to the protein secondary structure [22]. The bands located at 1003 cm−1 and 1032 cm−1 are associated with the aromatic side chains of phenylalanine [23]. A weak shoulder at 1174 cm−1 is assigned to the amino acid tyrosine in β-amyloid conformation [24]. A peak centred at 1445 cm−1 attributed to the bending vibrations of CH2 bond. A band detected at 1347 cm− 1 relates to Cα\C and C\H bonds. In addition, the peak at 1400 cm−1 is associated with the symmetric stretching of carboxyl group (COO−) side chain in aspartic acid. After contamination of the TiO2 surface with β-amyloid, a slight shift of ~5 cm−1 for the amide I band and ~15 cm−1 for the amide III band are observed. The Raman stretching mode for the C\COO− appeared at ~ 930 cm− 1 and 1390 cm−1 for the free β-amyloid, and following
is in the anatase phase. The results agree with those obtained by Sousa et al. [14]. 3.4. X-ray photoelectron spectroscopy Fig. 3 illustrates the high resolution XPS spectrum of the TiO2 thin film. The peak located at 464.7 eV can be assigned to Ti 2p1/2 and the peak located at 458.4 eV corresponds to Ti 2p3/2. The splitting between these peaks is around 5.8 eV, which is indicative of Ti+4 in the anatase phase of the TiO2 film. The deconvoluted Ti 2p3/2 spectrum confirmed two Gaussian components centred at 457.2 and 458.7 eV, and these were assigned to TiO and TiO2, respectively [18]. The quantitative analysis showed that the yield of Ti+4 signal was about 91% of the total titanium content. The O 1s core-level spectrum can be deconvoluted into three components. The lower binding energy centred at 530.7 eV corresponding to the lattice oxygen of TiO2. The second band located at 531.9 eV is assigned to physisorbed water or \OH groups on the surface in \Ti(OH)\O\Ti\(OH)\. The peak at 532.7 eV is related to oxygen in
800
C 1s
Intensity (cps)
Ti 2p
O1s
(A)
229
carbonyl group. In addition, the ratio of oxygen to titanium (O:Ti) was calculated from the XPS peak areas, and it was 1.86:1.0. These results correlate to previous analyses by XPS [19].
Ti-O (Eg)
Ti-O (A1g)
O-Ti-O (B1g)
O-Ti-O(Eg)
700
600
500
400
300
200
100
0
Binding energy (eV)
(C)
1
/ Ti 2p 2
466
464
Ti=O
Intensity (cps)
+4 Ti
+2 Ti
462
C=O
5.8 eV
468
Intensity (cps)
3
/ Ti 2p 2
(B)
460
Binding energy (eV)
458
456
534
O-H
Intensity (a/u)
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
532
530
Binding energy (eV)
Fig. 3. XPS survey scan of TiO2 (A) with peak fitting of Ti 2p (B) and O1s (C) bands.
528
230
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Fig. 4. AFM images of TiO2 before (A) and after (A*) annealing; β-amyloid on stainless steel (B) and β-amyloid on TiO2 (C) before, and after exposure to UV-B (B* and C*).
adsorption onto TiO2, this band is shifted to 953 cm−1 and 1403 cm−1 [25]. The broad band at 1280 cm−1 probably contains some contribution from amide III. 3.6.2. Curve fitting analysis of amid I band Fig. 6 illustrates the curve fitting for amide I bands for free β-amyloid (non-adsorbed), and β-amyloid adsorbed on TiO2, before and after irradiation with UVB. The key components are given in Table 1. The essential spectral components of the amide I band are the random-coil structure (1634–1638 cm−1), the α-helix (1650–1655 cm−1), the βsheet (1665–1678 cm−1) and the β-turn (1685–1696 cm−1) [18,26]. A broad band at ~1613 cm−1 can be attributed to aromatic ring vibrations of both phenylalanine and tyrosine residues [27]. Quantitative analysis of the amide I band revealed that free βamyloid (non-adsorbed) possesses both α-helix and β-sheet band with significant level of random coil. A band centred at 1633 cm− 1 with ~10% intensity is related to the random coil. A peak was found at 1652 cm−1 with 36% intensity which is attributed to α-helix conformation and ~34% of the band intensity is observed at 1667 cm−1 which is typically assigned to a β-sheet structure. The last peak observed at 1684 cm−1, with 11% intensity returns to the beta strand or polyprolin (PPII) conformation (Table 1). These results are comparable to those obtained by Schmechel et al. [28].
In the case of β-amyloid adsorbed on TiO2, the Raman amide I band narrows significantly and the peak maximum shifted to 1672 cm−1, whilst the beta strand content of the peptide is preserved (13%). A substantial increase in the β-sheet band at 1669 cm−1 from 34% to 69% is observed. On the other hand, the intensity of α-helix content dramatically decreases to less than 8%, along with the disappearance of random coil structure. The conformational transitions of α-helix, and random coil to β-sheet structure are accompanied by the formation of amyloid aggregates (fibrils) [29,30]. These results indicate that TiO2 surface leads to increase the rate of amyloid fibril formation. Following exposure of the contaminated TiO2 to UV-B, the secondary structures of amide I band slightly shifted towards a higher frequency (1670 cm−1) when compared with free β-amyloid. As a result of UVB treatment, changes in the secondary structural elements of β-amyloid were observed. A significant decrease in α-helical content (~ 28%) with a slight increase in β-sheet structure (38%) was found (Table 1). On the other hand, the transition of random coil component (1633 cm−1) to β-sheet (~1669 cm−1) was not observed following UV irradiation. Interestingly, a significant red shift of aromatic ring band from 1612 cm−1 to 1591 cm−1 is found, which is due to tryptophanyl and tyrosinyl radicals, which can be generated during UV treatment. Tryptophan radicals play a significant role in mediating biological electron transfer and catalytic processes [31]. Perhaps, the photo-oxidation of
Amide I
Amide III
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Intensity (a/u)
(E)
(D)
(C)
(B)
1000
1200
1400
1600
(A)
1800
2000
Raman Shift (cm-1) Fig. 5. Raman spectra of; free β-amyloid (β-A) (A), β-A on stainless steel (B), β-A on TiO2 (C), β-A on stainless steel following UV-B irradiation for 120 min (D), and β-A on TiO2 treated by UV-B irradiation for 120 min (E).
aromatic amino acids might lead to a side chain modification leading to conformational change and thus β-amyloid becomes amorphous aggregation [32]. These results agree well with AFM data and indicate that UV irradiation of the β-amyloid adsorbed on TiO2 causes accumulation of smaller β-amyloid aggregates. The UVB irradiation of the β-amyloid adsorbed on TiO2 resulted in a relative change in the conformation of the peptide from β-sheet to α-helix and therefore, photocatalysis could inhibit or reduce the aggregation of β-amyloid into fibrils.
The amide I band is more sensitive to conformational changes. The relative quantity of β-amyloid peptide that remained on the surface after UV-B exposure was estimated using a calibration curve based on Raman intensity and band area of amide I (Fig. 7). Interestingly, it was found that UV-B irradiation was quite effective in removing the peptide in both samples (stainless steel and TiO2). The data from the TiO2 films clearly demonstrated that amide I band was reduced by 26 ± 3.1% after 30 min of sample irradiation, whilst after 2 h of UV-B light irradiation, the amide I band ratio decreased by 64 ± 3.4%. The amide I peak area of amyloid on the stainless steel was decreased by 9% and 27% at 30 and 120 min of UV irradiation, respectively. The analysis data obtained from the t-test showed that there was a statistically significant difference (t = 14.8, p = 0.001) between the two groups of contaminated amyloid samples (TiO2 and stainless steel) after treatment with UV-B. This difference in value between stainless steel and TiO2 is due to the photocatalytic activity of TiO2. The denaturation of the peptide may be due to the disruption of the hydrogen bond network in β-amyloid, because the peptide chains are stabilized by hydrogen bonds between atoms of the amino acid backbones to adjacent parallel chains [33].
3.6.3. Atomic force microscopy (AFM) of β-amyloid The AFM topography images of the β-amyloid layer deposited on the surfaces are shown in Fig. 4. The root mean square (Rq) and mean roughness (Ra) values of amyloid on the stainless steel are around 9.3 nm and 7.3 nm, respectively (Table 2). Following the exposure of βamyloid on stainless steel to UV-B irradiation for 120 min (light control) the Rq and Ra values were changed to 6.6 nm and 5.3 nm, respectively. The analysis of AFM data for β-amyloid on TiO2 demonstrated that the Rq and Ra roughness average parameters were 7.8 nm and 6.1 nm,
(B) Intensity (a.u)
(A) Intensity (A.U)
231
1652 1667
1610
1672
1635 1685
1686
1616 1652
1580 1600 1620 1640 1660 1680 1700
1580
Raman Shift (cm-1)
1600
1620
1640
1660
1680
1700
Raman shift (cm-1)
Intensity (a.u)
(C)
1669
1654 1591
1580
1600
1684
1633
1620
1640
1660
1680
1700
Raman Shift (cm-1) Fig. 6. Curve-fitting analysis of the amide I in Raman spectra of: free β-amyloid (A), and β-A on TiO2 before (B) and after (C) UVB irradiation.
232
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Table 1 Raman spectra analysis of amide I band of β-amyloid by curve fitting. Free β-amyloid
Amide I sub-bands
ν (cm−1) Aromatic Random coil α-Helix β-Sheet PPII
β-Amyloid on TiO2
Band (%)
1613 1633 1652 1667 1684
Table 2 Roughness values (rms (Rq) and Ra) of TiO2 surface with and without β-amyloid contamination and before and after UV-B light irradiation.
9.4 9.6 36.1 33.6 11.2
Before UV-B irradiation
After UV-B irradiation
ν (cm−1)
ν (cm−1)
Band (%)
1610
9.9
1653 1672 1685
7.7 68.9 13.4
1591 1633 1653 1669 1687
Samples
Band (%) TiO2 β-Amyloid on stainless steel β-Amyloid on TiO2
10.6 8.9 27.8 38.4 14.2
respectively. One can conclude that adsorption of β-amyloid on TiO2 enhanced the fibrillation. Whilst after exposure to UV-B light both Rq and Ra values were significantly decreased compared to that before UV irradiation and the values were 4.1 and 3.2 nm, respectively (Table 1). The flattened globular shape of β-amyloid on TiO2 can be rationalized by the amphipathic character of the peptide. The affinity of β-amyloid towards the surface of TiO2 is undoubtedly driven by aspartic, glutamine and serine which are located in the hydrophilic block (1–25), because contact of hydrophobic block (25–40) with the surface of TiO2 is unfavourable energetically [34]. In addition the flattened shape of β-amyloid after being exposed to UV-B light may be accompanied by considerable change in the conformation of contaminated β-amyloid under the effect of photocatalytic activity of TiO2 surface [35]. AFM imaging of β-amyloid on TiO2 revealed that no globular aggregates and no fibrils are observed. The results are in good agreement with the experimental data obtained by Arimon et al. [36].
3.6.4. X-ray photoelectron spectroscopy (XPS) of β-amyloid The XPS survey scans of β-amyloid layer on the TiO2 surface showed significant changes in the carbon content at 284.7 eV with full width half maximum (FWHM) of ~1.54 eV and oxygen level at 530 eV with FWHM of ~ 1.21 eV; in addition, a new peak was observed at ~ 400 eV with FWHM of 1.32 eV, which belongs to nitrogen (N1s) (Fig. 8). A Gaussian line shape was used for the curve-fits, and the width of the individual peaks in the curve fits for the C1s, O1s and N1s spectra was constrained to a maximum of 0.8 eV, 1.1 eV and 0.9 eV, respectively.
100 (A)
(B)
Rq (nm)
Ra (nm)
Rq (nm)
Ra (nm)
23.2 ± 2.7 9.3 ± 1.3 7.8 ± 1.2
17.8 ± 2.1 7.3 ± 1.2 6.1 ± 1.0
6.6 ± 1.1 4.1 ± 0.6
5.3 ± 0.7 3.2 ± 0.4
The XPS C1s spectrum of β-amyloid on TiO2 has been deconvoluted into three contributions corresponding to well identified carbon bonds present in the protein. The first component centred at 285.0 eV is related to aliphatic carbons of the amino acid pending groups such as C\C, C_C, and C\H. The band denoted at 286.4 eV is attributed to C\(N, O) single bonds of the protein backbone. The band located at 288.5 eV is assigned to O_C\O (carboxyl group) and O_C\N from peptide bonds [37]. Following UVB irradiation of the β-amyloid adsorbed on TiO2 the XPS C1s spectrum was deconvoluted into five sub peaks centred at; 285.1 eV, 286.4 eV, 287.7 eV (CO), 289.0 eV (peptide bond) and the band that appeared at 292.6 eV corresponding to free carboxyl group (\COO−). There is evidence for conformational changes in β-amyloid with production of free carboxyl acid. The rate of degradation of the β-amyloid was greater on the TiO2 surface as compared to the stainless steel surface due to the photocatalytic activity of the TiO2 (Fig. 8). Furthermore, the results correlate well with the Raman studies in which the amide I band intensity was reduced following UV-B irradiation. The XPS N1s core level spectra show a strong peak at ~400 eV, which is attributed to the presence of β-amyloid attached on the surface of the samples. Three de-convoluted peaks were observed from band fitted spectra of the N1s; the first component centred at 399.9 eV (68%) which is attributed to (C\N), a peak located at 401.6 eV (21%) for N\CO and free amino groups (NH2), and the last signal at 402.8 eV (11%) which corresponded to positively charged amino group (NH+ 3 ) [38]. After exposure to UVB, the (NH+ 3 ) band area increased to 17% whilst the (NH2) peak area decreased to 14%; both peaks are slightly shifted towards higher energy. The XPS O1s spectrum is considerably broader after amyloid contamination on TiO2 surface (Fig. 7). The deconvolution of the O1s spectrum gives three sub peaks. The band centred at 531.4 eV is related to the oxygen present in the peptide chain of the β-amyloid. A component located at 532.2 eV is generated by the overlapping between the \OH groups of the TiO2 and the peptide oxygen of the β-amyloid. The signal at 533.3 eV corresponds to the carbonyl oxygen of the ester [39].
(C)
60
40
Raman intensity of Amide I (a.u)
% of amide I band
After exposure to UV-B
Rq or root mean square (rms) roughness is based on a least square calculated with the best fit of the height points. R a is obtained by a logarithm which measures the average deviation between the peaks and the values from the mean line of the surface. ± represents the values calculated and standard deviation (three images).
ν: Raman shift band (cm−1).
80
Before exposure to UV-B
4. Conclusions 320 280 240 200 160 120 80 40 0 0
10
20
30
40
(D)
50
quantity amount of β−amyloid (μg/ml)
0
20
40
60
80
100
120
UV irradiation time (min.) Fig. 7. The determination of β-amyloid (β-A) concentration based on amide I band intensity; β-A on stainless steel (A) and β-A on TiO2 (B) before UV treatment, and β-A on stainless steel (C) and β-A on TiO2 (D) following UV irradiation. The inset plot shows the obtained calibration curve of amide I. n = 5 and error bar shows the standard deviation.
TiO2 thin films were deposited on stainless steel using magnetron sputter deposition. After annealing at 430 °C for 3 h, the film structure and surface morphology were characterised by XRD, AFM and XPS. The results suggested that TiO2 thin film was mainly anatase with no rutile phase. The adsorption of β-amyloid protein was investigated using AFM, Raman and XPS analyses. The effect of UVB irradiation was analysed by the changes in the AFM, Raman and XPS analyses. Upon exposure to UVB, a reduction in the intensity of the amide I of β-amyloid band is observed. Furthermore, after 120 min of irradiation of amyloid on TiO2, the amide I band was reduced by 72%. TiO2 is well known for its photocatalytic activity where UV excitation yields electron/hole pairs which react at the surface to give reactive oxygen species including hydroxyl radical and superoxide radical anion. The degradation of
233
N 1s
Intensity (cps)
C 1s
O 1s
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Ti2p
β-amyloid on TiO2
600
450
300
150
0
Binding energy (eV)
Intensity (cps)
(A*)
Intensity (cps)
(A)
C-C C=C
C-O C-N
C-N
peptide bond
294
292
290
288
286
284
282
294
−
292
C-O
peptide
COO
290
288
286
284
282
Binding energy (eV)
Binding energy eV
C-N
C-N
Intensity (cps)
(B*)
Intensity (cps)
(B)
N-H
N-H
406
404
+ NH3
+ NH3
408
C-C C=C
402
400
398
396
408
406
Binding energy (eV)
404
402
400
398
396
394
Binding energy (eV)
Intensity (cps)
(C*)
Intensity (a/u)
(C)
CO
CO
OH
OH
C=O -O-
534
C=O -O-
531
528
Binding Energy (eV)
536
534
532
530
528
Binding Energy (eV)
Fig. 8. XPS survey scan of β-amyloid onto TiO2, with peak fitting of C1s (A), N1s (B) and O1s (C) before and (A*), (B*), (C*) after treatment by UV-B irradiation.
the protein on the surface is due to a combination of photolysis and photocatalysis. From AFM imaging, the average height of degraded amyloid remained in the range of 5–6 nm. The amyloid degradation on TiO2 has been visualized and suggested to be the result of the formation of
pseudomicellar structures of an amphipathic molecule such as βamyloid on a hydrophilic substrate. UV irradiation of surfaces may be an effective route to the decontamination of medical devices which have a TiO 2 coating e.g. titanium implants, and TiO2 films
234
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
may be utilised as photocatalytic coatings for the decontamination of other medical devices. Acknowledgements This work was supported by SFI under the investigator programme, Award No. 05/IN.1/B30/NS. The authors would like to acknowledge, all staff in the Nanotechnology Integrated Bio-Engineering Centre (NIBEC), University of Ulster, and National Centre for Sensor Research (NCSR), Dublin City University, for assistance with the analysis. References [1] S. Aulic, M.L. Bolognesi, G. Legname, Small-molecule theranostic probes: a promising future in neurodegenerative diseases, Int. J. Cell Biol. (2013). http://dx.doi.org/10. 1155/2013/150952 (Article ID 150952, 19 pages). [2] I.P. Lipscomb, A.K. Sihota, C.W. Keevil, Comparison between visual analysis and microscope assessment of surgical instrument cleanliness from sterile service departments, J. Hosp. Infect. 68 (2008) 52–58. [3] J.A. Cam, G. Bu, Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family, Mol. Neurodegener. 1 (2006) 1–13. [4] A.L. Biere, B. Ostaszewski, E.R. Stimson, B.T. Hyman, J.E. Maggio, D.J. Selkoe, Amyloid β-peptide is transported on lipoproteins and albumin in human plasma, J. Biol. Chem. 271 (51) (1996) 32916–32922. [5] W.-Q. Zou1, X. Xiao, J. Yuan, G. Puoti, H. Fujioka, X. Wang, S. Richardson, X. Zhou, R. Zou, S. Li, X. Zhu, P.L. McGeer, J. McGeehan, G. Kneale, D.E. Rinco-Limas, P. Fernandez-Funez, H-g. Lee, M.A. Smith, R.B. Petersen, J.-P. Guo, Amyloid beta interacts mainly with insoluble prion protein in the Alzheimer brain, J. Biol. Chem. 286 (2011) 15095–15105. [6] D.M. Taylor, Inactivation of prions by physical and chemical means, J. Hosp. Infect. 43 (1999) S69–S76. [7] M.G. Tyshenkoa, S. ElSaadanyb, T. Orabya, S. Darshana, W. Aspinallcd, R. Cookee, A. Catfordb, D. Krewskiaf, Expert elicitation for the judgment of prion disease risk uncertainties, J. Toxic. Environ. Health A 74 (2011) 261–285. [8] C.S. Atwood, R.N. Martins, M.A. Smith, G. Perry, Senile plaque composition and posttranslational modification of amyloid-[beta] peptide and associated proteins, Peptides 23 (7) (2002) 1343–1350. [9] A.W. Bryan, M. Menke, L.J. Cowen, S.L. Lindquist, B. Berger, BETASCAN: probable βamyloids identified by pairwise probabilistic analysis, PLoS Comput. Biol. 5 (2009) e1000333. [10] B.B. Zhang, Y.F. Zheng, Y. Liu, Effect of Ag on the corrosion behavior of Ti–Ag alloys in artificial saliva solutions, Dent. Mater. 25 (2009) 672–677. [11] M. Aziz-Kerrzo, K.G. Conroy, A.M. Fenelon, S.T. Farrell, C.B. Breslin, Electrochemical studies on the stability and corrosion resistance of titanium-based implant materials, Biomaterials 22 (12) (2001) 1531–1539. [12] X. Liu, P.K. Chu, C. Ding, Surface modification of titanium, titanium alloys, and related materials for biomedical applications, Mater. Sci. Eng. R 47 (2004) 49–121. [13] M. Niinomi, Recent metallic materials for biomedical applications, Met. Mater. Trans. A 32A (2001) 477–486. [14] S.R. Sousa, P.M. Ferreira, B. Saramago, L.V. Melo, M.A. Barbosa, Human serum albumin adsorption on TiO2 from single protein solutions and from plasma, Langmuir 20 (22) (2004) 9745–9754. [15] P.S.M. Dunlop, T.A. McMurray, J.W.J. Hamilton, J.A. Byrne, Photocatalytic inactivation of Clostridium perfringens spores on TiO2 electrodes, J. Photochem. Photobiol. A Chem. 196 (2008) 113–119. [16] I. Paspaltsis, C. Berberidou, I. Poulios, T. Sklaviadis, Photocatalytic degradation of prions using the photo-Fenton reagent, J. Hosp. Infect. 71 (2009) 149–156. [17] S. Kumar, N.K. Verma, M.L. Singla, Size dependent reflective properties of TiO2 nanoparticles and reflectors made thereof, Dig. J. Nanomater. Bios. 7 (2012) 607–619.
[18] O. Akhavan, E. Ghaderi, Capping antibacterial Ag nanorods aligned on Ti interlayer by mesoporous TiO2 layer, Surf. Coat. Technol. 203 (2009) 3123–3128. [19] M.H. Ahmed, T. Keyes, J. Byrne, C.W. Blackledge, J.W. Hamilton, Adsorption and photocatalytic degradation of human serum albumin on TiO2 and Ag–TiO2 films, J. Photochem. Photobiol. A Chem. 222 (2011) 123–131. [20] R. Hazan, S. Sreekantan, A.A. Khalil, I.M.S. Nordin, I. Mat, Surface engineering of titania for excellent fibroblast 3T3 cell–metal interaction, J. Phys. Sci. 20 (2007) 35–47. [21] C.S. Atwood, V.E. Anderson, S.L. Siedlak, M.A. Smith, G. Perry, P.R. Carey, Metal binding and oxidation of amyloid-β within isolated senile plaque cores: Raman microscopic evidence, Biochemistry 42 (2003) 2768–2773. [22] W.K. Surewicz, E.M. Jones, A.C. Apetri, The emerging principles of mammalian prion propagation and transmissibility barriers: insight from studies in vitro, Acc. Chem. Res. 39 (9) (2006) 654–662. [23] I. Choi, Y.S. Huh, D. Erickson, Ultra-sensitive, label-free probing of the conformational characteristics of amyloid beta aggregates with a SERS active nanofluidic device, Microfluid. Nanofluid. 12 (2012) 663–669. [24] Z. Kristofikova, V. Kopecky, K. Hofbauerova, P. Hovorkova, D. Rpova, Complex of amyloid-β peptides with 24-hydroxycholesterol and its effect on hemicholinium3 sensitive carriers, Neurochem. Res. 33 (2008) 412–421. [25] S. Stewart, P.M. Fredericks, Surface-enhanced Raman spectroscopy of peptides and proteins adsorbed on an electrochemically prepared silver surface, Spectrochim. Acta A 55 (1999) 1615–1640. [26] M.A. Strehle, P. Rosch, R. Petry, A. Hauck, R. Thull, W. Kiefer, J. Popp, Phys. Chem. Chem. Phys. 6 (2004) 5232–5236. [27] C. Gullekson, L. Lucas, K. Hewitt, L. Kreplak, Surface-sensitive Raman spectroscopy of collagen I fibrils, Biophys. J. 100 (2011) 1837–1845. [28] A. Schmechel, H. Zentgraf, S. Scheuermann, G. Fritz, R. Pipkorn, J.r Reed, K. Beyreuther, T.A. Bayer, G. Multhaup, Alzheimer β-amyloid homodimers facilitate Aβ-fibrillization and the generation of conformational antibodies, J. Biol. Chem. 278 (2003) 35317–35324. [29] C. Zhang, Y. Liu, J. Gilthorpe, J.R.C. van der Maarel, MRP14 (S100A9) protein interacts with Alzheimer beta-amyloid peptide and induces its fibrillization, PLoS ONE 7 (3) (2012) e32953. [30] I.-H. Chou, M. Benford, H.T. Beier, G.L. Cote, Nanofluidic biosensing for β-amyloid detection using surface enhanced Raman spectroscopy, Nano Lett. 8 (6) (2008) 1729–1735. [31] H.S. Shafaat, B.S. Leigh, M.J. Tauber, J.E. Kim, Resonance Raman characterization of a stable tryptophan radical in an azurin mutant, J. Phys. Chem. B 113 (2009) 382–388. [32] A.K. Thakur, C.M. Rao, UV-light exposed prion protein fails to form amyloid fibrils, PLoS ONE 3 (7) (2008) e2688. [33] C.L. Worth, T.L. Blundell, On the evolutionary conservation of hydrogen bonds made by buried polar amino acids: the hidden joists, braces and trusses of protein architecture, BMC Evol. Biol. 10 (2010) 161. [34] T. Kowalewski, D.M. Holtzman, In situ atomic force microscopy study of Alzheimer's β-amyloid peptide on different substrates: new insights into mechanism of β-sheet formation, Proc. Natl. Acad. Sci. U.S.A. Biophys. 96 (1999) 688–3693. [35] M. Lysetska, A. Knoll, D. Boehringer, T. Hey, G. Krauss, G. Krausch, UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study, Nucleic Acids Res. 30 (2002) 2686–2691. [36] M. Arimon, I. D-Perez, M.J. Kogan, N. Durany, E. Giralt, F. Sanz, X. F-Busquets, Fine structure study of Aß1–42 fibrillogenesis with atomic force microscopy, J. Fed. Am. Soc. Exp. Biol. 19 (2005) 1344–1346. [37] Y. Ohko, K. Ichiroiuchi, C. Niwa, T. Tatsuma, T. Nakashima, T. Iguchi, Y. Kubota, A. Fujishima, 17β-Estradiol degradation by TiO2 photocatalysis as a means of reducing estrogenic activity, Environ. Sci. Technol. 36 (2002) 4175–4181. [38] R. Flamia, G. Lanza, A.M. Salvi, J.E. Castle, A.M. Tamburro, Conformational study and hydrogen bonds detection on elastin-related polypeptides using X-ray photoelectron spectroscopy, Biomacromolecules 6 (2005) 1299–1309. [39] M.H. Ahmed, T.E. Keyes, J.A. Byrne, The photocatalytic inactivation effect of Ag–TiO2 on β-amyloid peptide (1–42), J. Photochem. Photobiol. A Chem. 254 (2013) 1–11
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Investigation of the inhibitory effects of TiO2 on the β-amyloid peptide aggregation Mukhtar H. Ahmed a,b,⁎, John A. Byrne b, Tia E. Keyes a a b
School of Chemical Science, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast, UK
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 12 January 2014 Accepted 1 March 2014 Available online 12 March 2014 Keyword: β-Amyloid peptide TiO2 XPS Raman spectroscopy Photocatalytic
a b s t r a c t TiO2 thin films are of great interest as biocompatible coatings and also as photocatalytic self-cleaning and antimicrobial coatings. In this work we used β-amyloid as a model for infectious protein to investigate the attachment and photocatalytic degradation. TiO2 films were prepared on stainless steel substrates using magnetron sputtering. The films were characterised before and after exposure to β-amyloid (1–42), using XRD, Raman spectroscopy, XPS and AFM. The TiO2 film was mostly composed of the anatase phase with a relatively high surface roughness. The presence of Raman peaks at 1668 cm−1 and 1263 cm−1, with the XPS spectral feature for nitrogen at 400 eV, confirmed the adsorption of amyloid on surface. Following exposure of the β-amyloid contaminated TiO2 to UV-B irradiation a slight shift of amide modes was observed. Furthermore, the amide I spectra show an overall decrease in α-helix content with presence of a minor peak around 1591 cm−1, which is related to tryptophanyl and tyrosinyl radicals, which can lead to conformational change of β-amyloid. The C1s band at 292.2 eV suggests the formation of free carboxylic acid. The loss in the crucial structure of β-amyloid leads to reduce the fibril formation, thought to be induced through a photocatalytic process. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Prion diseases, known as transmissible spongiform encephalopathies (TSEs), occur in both humans (Creutzfeldt–Jakob disease (CJD)) and animals (e.g. bovine spongiform encephalopathy (BSE)). Prion diseases are a group of rapidly progressive disorders characterised by a defined spectrum of clinical abnormalities. The infectious agent consists merely of protein and is capable of replicating and transmitting infection without the need for informational nucleic acids. There has been interest in proteins causing neurodegeneration since they may all act as prions (such as β-amyloid and α-synuclein) [1]. The prion-causing agent is highly robust and has been shown to remain infectious even after modern cleaning and inactivation regimes such as autoclaving (121 °C, 30 min) or chemical methods, e.g. formaldehyde gas, have been implemented [2]. β-Amyloid (Aβ) with ~4 kDa is the major component of the amyloid plaques found in prion diseases [3]. β-Amyloid is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions (1.0 nM) [4]. The β-amyloid peptides can aggregate into soluble oligomers or fibrillar assembles with the tinctoral properties of amyloids [5]. The β-amyloid structure is extremely stable and accumulates in infected tissue (nerve cells), causing tissue damage and cell death. This ⁎ Corresponding author at: Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast, UK. E-mail address: [email protected] (M.H. Ahmed).
http://dx.doi.org/10.1016/j.msec.2014.03.011 0928-4931/© 2014 Elsevier B.V. All rights reserved.
stability means that prions are resistant to denaturation by chemical and physical agents, difficult to remove and or degrade, making disposal and containment of these proteins difficult [6]. Therefore, these diseases can be transmitted, as adverse effects of medical treatment (iatrogenic transfer) and have occurred when contaminated surgical instruments have been reused [7]. Furthermore, many medical devices cannot be effectively sterilized with classical sterilization methods due to damage by heat or chemical disinfectants [8], and such techniques are often ineffective in decontamination of potentially infectious proteins [9]. Therefore, alternative methods of surface decontamination are required. Titanium and titanium alloys are the most widely used in medical applications, because of their excellent properties including chemical stability, low density, high strength, corrosion resistance, and biocompatibility [10–12]. In-vitro and in-vivo experiments have demonstrated that titanium dioxide (TiO2) coatings exhibit good blood compatibility, and do not appear to induce platelet aggregation, damage red blood cells or adsorb fibrin strongly [13,14]. TiO2 under UV excitation exhibits photocatalytic activity wherein UV excitation promotes electrons to the conduction band creating holes in the valence band. These charge carriers can migrate to the surface and take part in redox reactions with water and molecular oxygen leading to the formation of reactive oxygen species including hydroxyl radical and superoxide radical anion. TiO2 photocatalysis has been reported to be effective for the inactivation of a wide range of pathogenic microorganisms implicated in Healthcare Associated Infections [15].
228
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
The photocatalytic degradation of proteins involves several chemical and photocatalytic stages and a great number of intermediates. Previous studies have proposed mechanisms of oxidative cleavage mediated by hydroxyl radical [OH•], which are able to attack the main and the side chains of proteins, producing molecules with lower molecular weight and finally CO2 [16]. In this work, stainless steel was coated with thin films of TiO2, followed by contamination of the surface with β-amyloid polypeptide. The photocatalytic denaturation of the β-amyloid under ultraviolet irradiation has been explored. Both adsorption and decontamination are examined using advanced surface analytical techniques, including Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). 2. Materials and methods 2.1. Preparation of TiO2 thin film Polished coupons of 316 stainless steel [48 samples of 2 × 2 cm2] were ultrasonically cleaned with acetone for 15 min, then washed using distilled water and dried with nitrogen gas. TiO2 thin film was then deposited on the coupons by radio-frequency (rf) magnetron sputtering using an unbalanced magnetron system (TEER UDP 450). Sputtering was carried out in an argon and oxygen plasma, with a Ti target (Teer, 99.6% pure) and pressure b5 × 10−6 Pa. The deposition process was carried out in the power regulation mode at 400 W, resulting in 0.98 A applied to the target. These conditions were optimised to give a maximum deposition rate. The partial pressures of reactive gas (oxygen) and working gas (argon) were controlled by the oxygen and argon flow rates. To deposit a stoichiometric film, a ratio of 15.0 cm3/min argon to 5.0 cm3/min oxygen giving 25% oxygen, was used. The deposition time was 8 h. To improve the crystallinity of the TiO2 film and obtain the anatase phase, samples were annealed at elevated temperatures in air (samples were heated at a rate of 5 °C/min to a temperature of 430 °C, held for 3 h, and cooled at 5 °C/min).
7 accumulations. This process was repeated at five different spots across the samples of TiO2 to assess the uniformity of the film. The film thickness was determined using Stylus profilometry (Dektak 8 Advanced Stylus profiler Veeco Instruments Inc., USA), automatic levelling was selected and system-based software obtained average step height calculation. Atomic force microscopy (AFM) was performed using the Digital Instruments, Veeco BioScope II. Imaging was performed under ambient conditions at 25 °C using a sharp silicon probe (R = 20 nm), attached to a low stress soft silicon nitride (SiN) cantilever with no reflecting coating on its back side [spring constant ~0.033 N/m and resonant frequency around 15 kHz and applied force of 500 mV]. The AFM exhibited a maximum scan area of 10 × 10 μm2 and a vertical range of 3 μm and was calibrated using calibration gratings purchased from Micro Masch. XPS analysis was carried out using an AXIS Ultra XPS system using a monochromatic Al-Kα X-ray source (hv 1486 eV) generated from an aluminium anode operating at an emission voltage of 15 kV with current of 5 mA at the source. The base pressure within the spectrometer during examination was 1.0 × 10−10 bar. 3. Results and discussion 3.1. TiO2 film thickness After annealing at 430 °C for 3 h, the film thickness of TiO2 samples has been determined and the values were around 100 ± 5 nm, yielding a film deposition rate of 12 nm/h. 3.2. X-ray diffraction (XRD) of TiO2 Fig. 1 shows the X-ray diffraction (XRD) analysis of TiO2 thin film, after the annealing process. The major peaks were detected at 2θ values of 25.3°, 37.9°, 48.2°, 53.0°, 55.9° and 62.6° which are corresponding to (101), (004), (200), (105), (211) and (204) anatase reflections of TiO2 respectively. The results are comparable to those obtained by Kumar et al. [17].
2.2. β-Amyloid contamination 3.3. Raman spectroscopy of TiO2
10
20
30
40
50
TiO (204) 2
TiO (105) 2
TiO (211) 2
A Bruker D8 X-ray diffractometer was used for XRD analysis. Diffraction patterns were collected in reflection-mode geometry from 20° to 80° 2θ at a rate of 0.05° 2θ/min. Raman spectra were recorded on a Horiba Jobin Yvon HR800UV microscope using ~2 mW from a 532 nm laser diode for excitation and the following parameters; confocal aperture 200 μm and spectral resolution ~ 5 cm− 1. A 100 × objective was employed and typical acquisition times were 5 s and that was repeated
TiO (200) 2
2.4. Film characterisation
TiO (004) 2
Following exposure to β-amyloid, samples were irradiated using UV-B, to determine the effect of UV photolysis and/or photocatalysis on the β-amyloid structure. The UV-B source was a fluorescent lamp (Philips, PLS 9W/12/2p) set ~ 6.0 cm above a Petri dish with peak emission at 315 nm. Dark and light control experiments were also undertaken. Dark control, samples were prepared and incubated under the same conditions but not exposed to light, by covering them with foil. Five samples were used for each time point within groups.
TiO (101) 2
2.3. UV-photocatalysis
The Raman spectrum of the TiO2 film after the annealing at 430 °C is shown in Fig. 2. The Raman active Ti\O stretching band is centred at 632 cm− 1 (Eg) and 518 cm− 1 (A1g). The bands at 400 cm− 1 and 155 cm−1, corresponded to both (B1g) and (Eg) bending vibrations of (O\Ti\O), respectively. The obtained data are consistent with anatase mode of TiO2. There was no evidence for the presence of rutile TiO2 which is characterised by modes at 606, 434 and 230 cm− 1. The Raman data correlates well with the XRD data, indicating that the TiO2
Intensity (a/u)
β-Amyloid (1–42) was obtained from Sigma Aldrich, and it was dissolved in 10 mM phosphate buffer at pH 7.4 to make a final concentration of ~ 1 × 10− 5 M and stored at − 30 °C. The samples were incubated with 50 μg/ml [~10 μM] of β-amyloid solution for 60 min at 37 ± 1 °C. At the end of the adsorption period, the samples were rinsed and washed using distilled water, and then dried.
60
2θ (degree) Fig. 1. X-ray diffraction (XRD) spectra of TiO2 thin film.
70
80
200
3.5. Atomic force microscopy of TiO2 thin film
400
600
800
The surface roughness of the TiO2 sample was characterised using AFM. Fig. 4 demonstrates the three dimensional morphology of TiO2. The annealed TiO2 film had a higher roughness than non-annealed film. The root mean square (Rq) roughness value of the deposited TiO2 film was around 23.2 nm ± 2.7. This finding is in agreement with the results published by Hazan et al. [20].
1000
Raman shift (cm-1)
3.6. Adsorption of β-amyloid onto surface of TiO2
Fig. 2. Raman spectra of TiO2 thin film.
3.6.1. Raman spectroscopy of β-amyloid Fig. 5 shows the Raman spectra of the β-amyloid adsorbed on the surface of TiO2, with and without exposure to UV-B irradiation. In the case of free β-amyloid (non-adsorbed), the vibration characteristic of the amide I band occurs at 1668 cm− 1, which involves mainly C_O stretching, C\N stretching, Cα\C\N bending, and N\H in-plane bending of α-helical peptide groups. Moreover, the amide III mode is a NH and C_O in plane bending modes with C\N stretch, and it is observed at 1263 cm− 1 [21]. The frequencies and shapes of these features are highly sensitive to the protein secondary structure [22]. The bands located at 1003 cm−1 and 1032 cm−1 are associated with the aromatic side chains of phenylalanine [23]. A weak shoulder at 1174 cm−1 is assigned to the amino acid tyrosine in β-amyloid conformation [24]. A peak centred at 1445 cm−1 attributed to the bending vibrations of CH2 bond. A band detected at 1347 cm− 1 relates to Cα\C and C\H bonds. In addition, the peak at 1400 cm−1 is associated with the symmetric stretching of carboxyl group (COO−) side chain in aspartic acid. After contamination of the TiO2 surface with β-amyloid, a slight shift of ~5 cm−1 for the amide I band and ~15 cm−1 for the amide III band are observed. The Raman stretching mode for the C\COO− appeared at ~ 930 cm− 1 and 1390 cm−1 for the free β-amyloid, and following
is in the anatase phase. The results agree with those obtained by Sousa et al. [14]. 3.4. X-ray photoelectron spectroscopy Fig. 3 illustrates the high resolution XPS spectrum of the TiO2 thin film. The peak located at 464.7 eV can be assigned to Ti 2p1/2 and the peak located at 458.4 eV corresponds to Ti 2p3/2. The splitting between these peaks is around 5.8 eV, which is indicative of Ti+4 in the anatase phase of the TiO2 film. The deconvoluted Ti 2p3/2 spectrum confirmed two Gaussian components centred at 457.2 and 458.7 eV, and these were assigned to TiO and TiO2, respectively [18]. The quantitative analysis showed that the yield of Ti+4 signal was about 91% of the total titanium content. The O 1s core-level spectrum can be deconvoluted into three components. The lower binding energy centred at 530.7 eV corresponding to the lattice oxygen of TiO2. The second band located at 531.9 eV is assigned to physisorbed water or \OH groups on the surface in \Ti(OH)\O\Ti\(OH)\. The peak at 532.7 eV is related to oxygen in
800
C 1s
Intensity (cps)
Ti 2p
O1s
(A)
229
carbonyl group. In addition, the ratio of oxygen to titanium (O:Ti) was calculated from the XPS peak areas, and it was 1.86:1.0. These results correlate to previous analyses by XPS [19].
Ti-O (Eg)
Ti-O (A1g)
O-Ti-O (B1g)
O-Ti-O(Eg)
700
600
500
400
300
200
100
0
Binding energy (eV)
(C)
1
/ Ti 2p 2
466
464
Ti=O
Intensity (cps)
+4 Ti
+2 Ti
462
C=O
5.8 eV
468
Intensity (cps)
3
/ Ti 2p 2
(B)
460
Binding energy (eV)
458
456
534
O-H
Intensity (a/u)
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
532
530
Binding energy (eV)
Fig. 3. XPS survey scan of TiO2 (A) with peak fitting of Ti 2p (B) and O1s (C) bands.
528
230
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Fig. 4. AFM images of TiO2 before (A) and after (A*) annealing; β-amyloid on stainless steel (B) and β-amyloid on TiO2 (C) before, and after exposure to UV-B (B* and C*).
adsorption onto TiO2, this band is shifted to 953 cm−1 and 1403 cm−1 [25]. The broad band at 1280 cm−1 probably contains some contribution from amide III. 3.6.2. Curve fitting analysis of amid I band Fig. 6 illustrates the curve fitting for amide I bands for free β-amyloid (non-adsorbed), and β-amyloid adsorbed on TiO2, before and after irradiation with UVB. The key components are given in Table 1. The essential spectral components of the amide I band are the random-coil structure (1634–1638 cm−1), the α-helix (1650–1655 cm−1), the βsheet (1665–1678 cm−1) and the β-turn (1685–1696 cm−1) [18,26]. A broad band at ~1613 cm−1 can be attributed to aromatic ring vibrations of both phenylalanine and tyrosine residues [27]. Quantitative analysis of the amide I band revealed that free βamyloid (non-adsorbed) possesses both α-helix and β-sheet band with significant level of random coil. A band centred at 1633 cm− 1 with ~10% intensity is related to the random coil. A peak was found at 1652 cm−1 with 36% intensity which is attributed to α-helix conformation and ~34% of the band intensity is observed at 1667 cm−1 which is typically assigned to a β-sheet structure. The last peak observed at 1684 cm−1, with 11% intensity returns to the beta strand or polyprolin (PPII) conformation (Table 1). These results are comparable to those obtained by Schmechel et al. [28].
In the case of β-amyloid adsorbed on TiO2, the Raman amide I band narrows significantly and the peak maximum shifted to 1672 cm−1, whilst the beta strand content of the peptide is preserved (13%). A substantial increase in the β-sheet band at 1669 cm−1 from 34% to 69% is observed. On the other hand, the intensity of α-helix content dramatically decreases to less than 8%, along with the disappearance of random coil structure. The conformational transitions of α-helix, and random coil to β-sheet structure are accompanied by the formation of amyloid aggregates (fibrils) [29,30]. These results indicate that TiO2 surface leads to increase the rate of amyloid fibril formation. Following exposure of the contaminated TiO2 to UV-B, the secondary structures of amide I band slightly shifted towards a higher frequency (1670 cm−1) when compared with free β-amyloid. As a result of UVB treatment, changes in the secondary structural elements of β-amyloid were observed. A significant decrease in α-helical content (~ 28%) with a slight increase in β-sheet structure (38%) was found (Table 1). On the other hand, the transition of random coil component (1633 cm−1) to β-sheet (~1669 cm−1) was not observed following UV irradiation. Interestingly, a significant red shift of aromatic ring band from 1612 cm−1 to 1591 cm−1 is found, which is due to tryptophanyl and tyrosinyl radicals, which can be generated during UV treatment. Tryptophan radicals play a significant role in mediating biological electron transfer and catalytic processes [31]. Perhaps, the photo-oxidation of
Amide I
Amide III
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Intensity (a/u)
(E)
(D)
(C)
(B)
1000
1200
1400
1600
(A)
1800
2000
Raman Shift (cm-1) Fig. 5. Raman spectra of; free β-amyloid (β-A) (A), β-A on stainless steel (B), β-A on TiO2 (C), β-A on stainless steel following UV-B irradiation for 120 min (D), and β-A on TiO2 treated by UV-B irradiation for 120 min (E).
aromatic amino acids might lead to a side chain modification leading to conformational change and thus β-amyloid becomes amorphous aggregation [32]. These results agree well with AFM data and indicate that UV irradiation of the β-amyloid adsorbed on TiO2 causes accumulation of smaller β-amyloid aggregates. The UVB irradiation of the β-amyloid adsorbed on TiO2 resulted in a relative change in the conformation of the peptide from β-sheet to α-helix and therefore, photocatalysis could inhibit or reduce the aggregation of β-amyloid into fibrils.
The amide I band is more sensitive to conformational changes. The relative quantity of β-amyloid peptide that remained on the surface after UV-B exposure was estimated using a calibration curve based on Raman intensity and band area of amide I (Fig. 7). Interestingly, it was found that UV-B irradiation was quite effective in removing the peptide in both samples (stainless steel and TiO2). The data from the TiO2 films clearly demonstrated that amide I band was reduced by 26 ± 3.1% after 30 min of sample irradiation, whilst after 2 h of UV-B light irradiation, the amide I band ratio decreased by 64 ± 3.4%. The amide I peak area of amyloid on the stainless steel was decreased by 9% and 27% at 30 and 120 min of UV irradiation, respectively. The analysis data obtained from the t-test showed that there was a statistically significant difference (t = 14.8, p = 0.001) between the two groups of contaminated amyloid samples (TiO2 and stainless steel) after treatment with UV-B. This difference in value between stainless steel and TiO2 is due to the photocatalytic activity of TiO2. The denaturation of the peptide may be due to the disruption of the hydrogen bond network in β-amyloid, because the peptide chains are stabilized by hydrogen bonds between atoms of the amino acid backbones to adjacent parallel chains [33].
3.6.3. Atomic force microscopy (AFM) of β-amyloid The AFM topography images of the β-amyloid layer deposited on the surfaces are shown in Fig. 4. The root mean square (Rq) and mean roughness (Ra) values of amyloid on the stainless steel are around 9.3 nm and 7.3 nm, respectively (Table 2). Following the exposure of βamyloid on stainless steel to UV-B irradiation for 120 min (light control) the Rq and Ra values were changed to 6.6 nm and 5.3 nm, respectively. The analysis of AFM data for β-amyloid on TiO2 demonstrated that the Rq and Ra roughness average parameters were 7.8 nm and 6.1 nm,
(B) Intensity (a.u)
(A) Intensity (A.U)
231
1652 1667
1610
1672
1635 1685
1686
1616 1652
1580 1600 1620 1640 1660 1680 1700
1580
Raman Shift (cm-1)
1600
1620
1640
1660
1680
1700
Raman shift (cm-1)
Intensity (a.u)
(C)
1669
1654 1591
1580
1600
1684
1633
1620
1640
1660
1680
1700
Raman Shift (cm-1) Fig. 6. Curve-fitting analysis of the amide I in Raman spectra of: free β-amyloid (A), and β-A on TiO2 before (B) and after (C) UVB irradiation.
232
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Table 1 Raman spectra analysis of amide I band of β-amyloid by curve fitting. Free β-amyloid
Amide I sub-bands
ν (cm−1) Aromatic Random coil α-Helix β-Sheet PPII
β-Amyloid on TiO2
Band (%)
1613 1633 1652 1667 1684
Table 2 Roughness values (rms (Rq) and Ra) of TiO2 surface with and without β-amyloid contamination and before and after UV-B light irradiation.
9.4 9.6 36.1 33.6 11.2
Before UV-B irradiation
After UV-B irradiation
ν (cm−1)
ν (cm−1)
Band (%)
1610
9.9
1653 1672 1685
7.7 68.9 13.4
1591 1633 1653 1669 1687
Samples
Band (%) TiO2 β-Amyloid on stainless steel β-Amyloid on TiO2
10.6 8.9 27.8 38.4 14.2
respectively. One can conclude that adsorption of β-amyloid on TiO2 enhanced the fibrillation. Whilst after exposure to UV-B light both Rq and Ra values were significantly decreased compared to that before UV irradiation and the values were 4.1 and 3.2 nm, respectively (Table 1). The flattened globular shape of β-amyloid on TiO2 can be rationalized by the amphipathic character of the peptide. The affinity of β-amyloid towards the surface of TiO2 is undoubtedly driven by aspartic, glutamine and serine which are located in the hydrophilic block (1–25), because contact of hydrophobic block (25–40) with the surface of TiO2 is unfavourable energetically [34]. In addition the flattened shape of β-amyloid after being exposed to UV-B light may be accompanied by considerable change in the conformation of contaminated β-amyloid under the effect of photocatalytic activity of TiO2 surface [35]. AFM imaging of β-amyloid on TiO2 revealed that no globular aggregates and no fibrils are observed. The results are in good agreement with the experimental data obtained by Arimon et al. [36].
3.6.4. X-ray photoelectron spectroscopy (XPS) of β-amyloid The XPS survey scans of β-amyloid layer on the TiO2 surface showed significant changes in the carbon content at 284.7 eV with full width half maximum (FWHM) of ~1.54 eV and oxygen level at 530 eV with FWHM of ~ 1.21 eV; in addition, a new peak was observed at ~ 400 eV with FWHM of 1.32 eV, which belongs to nitrogen (N1s) (Fig. 8). A Gaussian line shape was used for the curve-fits, and the width of the individual peaks in the curve fits for the C1s, O1s and N1s spectra was constrained to a maximum of 0.8 eV, 1.1 eV and 0.9 eV, respectively.
100 (A)
(B)
Rq (nm)
Ra (nm)
Rq (nm)
Ra (nm)
23.2 ± 2.7 9.3 ± 1.3 7.8 ± 1.2
17.8 ± 2.1 7.3 ± 1.2 6.1 ± 1.0
6.6 ± 1.1 4.1 ± 0.6
5.3 ± 0.7 3.2 ± 0.4
The XPS C1s spectrum of β-amyloid on TiO2 has been deconvoluted into three contributions corresponding to well identified carbon bonds present in the protein. The first component centred at 285.0 eV is related to aliphatic carbons of the amino acid pending groups such as C\C, C_C, and C\H. The band denoted at 286.4 eV is attributed to C\(N, O) single bonds of the protein backbone. The band located at 288.5 eV is assigned to O_C\O (carboxyl group) and O_C\N from peptide bonds [37]. Following UVB irradiation of the β-amyloid adsorbed on TiO2 the XPS C1s spectrum was deconvoluted into five sub peaks centred at; 285.1 eV, 286.4 eV, 287.7 eV (CO), 289.0 eV (peptide bond) and the band that appeared at 292.6 eV corresponding to free carboxyl group (\COO−). There is evidence for conformational changes in β-amyloid with production of free carboxyl acid. The rate of degradation of the β-amyloid was greater on the TiO2 surface as compared to the stainless steel surface due to the photocatalytic activity of the TiO2 (Fig. 8). Furthermore, the results correlate well with the Raman studies in which the amide I band intensity was reduced following UV-B irradiation. The XPS N1s core level spectra show a strong peak at ~400 eV, which is attributed to the presence of β-amyloid attached on the surface of the samples. Three de-convoluted peaks were observed from band fitted spectra of the N1s; the first component centred at 399.9 eV (68%) which is attributed to (C\N), a peak located at 401.6 eV (21%) for N\CO and free amino groups (NH2), and the last signal at 402.8 eV (11%) which corresponded to positively charged amino group (NH+ 3 ) [38]. After exposure to UVB, the (NH+ 3 ) band area increased to 17% whilst the (NH2) peak area decreased to 14%; both peaks are slightly shifted towards higher energy. The XPS O1s spectrum is considerably broader after amyloid contamination on TiO2 surface (Fig. 7). The deconvolution of the O1s spectrum gives three sub peaks. The band centred at 531.4 eV is related to the oxygen present in the peptide chain of the β-amyloid. A component located at 532.2 eV is generated by the overlapping between the \OH groups of the TiO2 and the peptide oxygen of the β-amyloid. The signal at 533.3 eV corresponds to the carbonyl oxygen of the ester [39].
(C)
60
40
Raman intensity of Amide I (a.u)
% of amide I band
After exposure to UV-B
Rq or root mean square (rms) roughness is based on a least square calculated with the best fit of the height points. R a is obtained by a logarithm which measures the average deviation between the peaks and the values from the mean line of the surface. ± represents the values calculated and standard deviation (three images).
ν: Raman shift band (cm−1).
80
Before exposure to UV-B
4. Conclusions 320 280 240 200 160 120 80 40 0 0
10
20
30
40
(D)
50
quantity amount of β−amyloid (μg/ml)
0
20
40
60
80
100
120
UV irradiation time (min.) Fig. 7. The determination of β-amyloid (β-A) concentration based on amide I band intensity; β-A on stainless steel (A) and β-A on TiO2 (B) before UV treatment, and β-A on stainless steel (C) and β-A on TiO2 (D) following UV irradiation. The inset plot shows the obtained calibration curve of amide I. n = 5 and error bar shows the standard deviation.
TiO2 thin films were deposited on stainless steel using magnetron sputter deposition. After annealing at 430 °C for 3 h, the film structure and surface morphology were characterised by XRD, AFM and XPS. The results suggested that TiO2 thin film was mainly anatase with no rutile phase. The adsorption of β-amyloid protein was investigated using AFM, Raman and XPS analyses. The effect of UVB irradiation was analysed by the changes in the AFM, Raman and XPS analyses. Upon exposure to UVB, a reduction in the intensity of the amide I of β-amyloid band is observed. Furthermore, after 120 min of irradiation of amyloid on TiO2, the amide I band was reduced by 72%. TiO2 is well known for its photocatalytic activity where UV excitation yields electron/hole pairs which react at the surface to give reactive oxygen species including hydroxyl radical and superoxide radical anion. The degradation of
233
N 1s
Intensity (cps)
C 1s
O 1s
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
Ti2p
β-amyloid on TiO2
600
450
300
150
0
Binding energy (eV)
Intensity (cps)
(A*)
Intensity (cps)
(A)
C-C C=C
C-O C-N
C-N
peptide bond
294
292
290
288
286
284
282
294
−
292
C-O
peptide
COO
290
288
286
284
282
Binding energy (eV)
Binding energy eV
C-N
C-N
Intensity (cps)
(B*)
Intensity (cps)
(B)
N-H
N-H
406
404
+ NH3
+ NH3
408
C-C C=C
402
400
398
396
408
406
Binding energy (eV)
404
402
400
398
396
394
Binding energy (eV)
Intensity (cps)
(C*)
Intensity (a/u)
(C)
CO
CO
OH
OH
C=O -O-
534
C=O -O-
531
528
Binding Energy (eV)
536
534
532
530
528
Binding Energy (eV)
Fig. 8. XPS survey scan of β-amyloid onto TiO2, with peak fitting of C1s (A), N1s (B) and O1s (C) before and (A*), (B*), (C*) after treatment by UV-B irradiation.
the protein on the surface is due to a combination of photolysis and photocatalysis. From AFM imaging, the average height of degraded amyloid remained in the range of 5–6 nm. The amyloid degradation on TiO2 has been visualized and suggested to be the result of the formation of
pseudomicellar structures of an amphipathic molecule such as βamyloid on a hydrophilic substrate. UV irradiation of surfaces may be an effective route to the decontamination of medical devices which have a TiO 2 coating e.g. titanium implants, and TiO2 films
234
M.H. Ahmed et al. / Materials Science and Engineering C 39 (2014) 227–234
may be utilised as photocatalytic coatings for the decontamination of other medical devices. Acknowledgements This work was supported by SFI under the investigator programme, Award No. 05/IN.1/B30/NS. The authors would like to acknowledge, all staff in the Nanotechnology Integrated Bio-Engineering Centre (NIBEC), University of Ulster, and National Centre for Sensor Research (NCSR), Dublin City University, for assistance with the analysis. References [1] S. Aulic, M.L. Bolognesi, G. Legname, Small-molecule theranostic probes: a promising future in neurodegenerative diseases, Int. J. Cell Biol. (2013). http://dx.doi.org/10. 1155/2013/150952 (Article ID 150952, 19 pages). [2] I.P. Lipscomb, A.K. Sihota, C.W. Keevil, Comparison between visual analysis and microscope assessment of surgical instrument cleanliness from sterile service departments, J. Hosp. Infect. 68 (2008) 52–58. [3] J.A. Cam, G. Bu, Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family, Mol. Neurodegener. 1 (2006) 1–13. [4] A.L. Biere, B. Ostaszewski, E.R. Stimson, B.T. Hyman, J.E. Maggio, D.J. Selkoe, Amyloid β-peptide is transported on lipoproteins and albumin in human plasma, J. Biol. Chem. 271 (51) (1996) 32916–32922. [5] W.-Q. Zou1, X. Xiao, J. Yuan, G. Puoti, H. Fujioka, X. Wang, S. Richardson, X. Zhou, R. Zou, S. Li, X. Zhu, P.L. McGeer, J. McGeehan, G. Kneale, D.E. Rinco-Limas, P. Fernandez-Funez, H-g. Lee, M.A. Smith, R.B. Petersen, J.-P. Guo, Amyloid beta interacts mainly with insoluble prion protein in the Alzheimer brain, J. Biol. Chem. 286 (2011) 15095–15105. [6] D.M. Taylor, Inactivation of prions by physical and chemical means, J. Hosp. Infect. 43 (1999) S69–S76. [7] M.G. Tyshenkoa, S. ElSaadanyb, T. Orabya, S. Darshana, W. Aspinallcd, R. Cookee, A. Catfordb, D. Krewskiaf, Expert elicitation for the judgment of prion disease risk uncertainties, J. Toxic. Environ. Health A 74 (2011) 261–285. [8] C.S. Atwood, R.N. Martins, M.A. Smith, G. Perry, Senile plaque composition and posttranslational modification of amyloid-[beta] peptide and associated proteins, Peptides 23 (7) (2002) 1343–1350. [9] A.W. Bryan, M. Menke, L.J. Cowen, S.L. Lindquist, B. Berger, BETASCAN: probable βamyloids identified by pairwise probabilistic analysis, PLoS Comput. Biol. 5 (2009) e1000333. [10] B.B. Zhang, Y.F. Zheng, Y. Liu, Effect of Ag on the corrosion behavior of Ti–Ag alloys in artificial saliva solutions, Dent. Mater. 25 (2009) 672–677. [11] M. Aziz-Kerrzo, K.G. Conroy, A.M. Fenelon, S.T. Farrell, C.B. Breslin, Electrochemical studies on the stability and corrosion resistance of titanium-based implant materials, Biomaterials 22 (12) (2001) 1531–1539. [12] X. Liu, P.K. Chu, C. Ding, Surface modification of titanium, titanium alloys, and related materials for biomedical applications, Mater. Sci. Eng. R 47 (2004) 49–121. [13] M. Niinomi, Recent metallic materials for biomedical applications, Met. Mater. Trans. A 32A (2001) 477–486. [14] S.R. Sousa, P.M. Ferreira, B. Saramago, L.V. Melo, M.A. Barbosa, Human serum albumin adsorption on TiO2 from single protein solutions and from plasma, Langmuir 20 (22) (2004) 9745–9754. [15] P.S.M. Dunlop, T.A. McMurray, J.W.J. Hamilton, J.A. Byrne, Photocatalytic inactivation of Clostridium perfringens spores on TiO2 electrodes, J. Photochem. Photobiol. A Chem. 196 (2008) 113–119. [16] I. Paspaltsis, C. Berberidou, I. Poulios, T. Sklaviadis, Photocatalytic degradation of prions using the photo-Fenton reagent, J. Hosp. Infect. 71 (2009) 149–156. [17] S. Kumar, N.K. Verma, M.L. Singla, Size dependent reflective properties of TiO2 nanoparticles and reflectors made thereof, Dig. J. Nanomater. Bios. 7 (2012) 607–619.
[18] O. Akhavan, E. Ghaderi, Capping antibacterial Ag nanorods aligned on Ti interlayer by mesoporous TiO2 layer, Surf. Coat. Technol. 203 (2009) 3123–3128. [19] M.H. Ahmed, T. Keyes, J. Byrne, C.W. Blackledge, J.W. Hamilton, Adsorption and photocatalytic degradation of human serum albumin on TiO2 and Ag–TiO2 films, J. Photochem. Photobiol. A Chem. 222 (2011) 123–131. [20] R. Hazan, S. Sreekantan, A.A. Khalil, I.M.S. Nordin, I. Mat, Surface engineering of titania for excellent fibroblast 3T3 cell–metal interaction, J. Phys. Sci. 20 (2007) 35–47. [21] C.S. Atwood, V.E. Anderson, S.L. Siedlak, M.A. Smith, G. Perry, P.R. Carey, Metal binding and oxidation of amyloid-β within isolated senile plaque cores: Raman microscopic evidence, Biochemistry 42 (2003) 2768–2773. [22] W.K. Surewicz, E.M. Jones, A.C. Apetri, The emerging principles of mammalian prion propagation and transmissibility barriers: insight from studies in vitro, Acc. Chem. Res. 39 (9) (2006) 654–662. [23] I. Choi, Y.S. Huh, D. Erickson, Ultra-sensitive, label-free probing of the conformational characteristics of amyloid beta aggregates with a SERS active nanofluidic device, Microfluid. Nanofluid. 12 (2012) 663–669. [24] Z. Kristofikova, V. Kopecky, K. Hofbauerova, P. Hovorkova, D. Rpova, Complex of amyloid-β peptides with 24-hydroxycholesterol and its effect on hemicholinium3 sensitive carriers, Neurochem. Res. 33 (2008) 412–421. [25] S. Stewart, P.M. Fredericks, Surface-enhanced Raman spectroscopy of peptides and proteins adsorbed on an electrochemically prepared silver surface, Spectrochim. Acta A 55 (1999) 1615–1640. [26] M.A. Strehle, P. Rosch, R. Petry, A. Hauck, R. Thull, W. Kiefer, J. Popp, Phys. Chem. Chem. Phys. 6 (2004) 5232–5236. [27] C. Gullekson, L. Lucas, K. Hewitt, L. Kreplak, Surface-sensitive Raman spectroscopy of collagen I fibrils, Biophys. J. 100 (2011) 1837–1845. [28] A. Schmechel, H. Zentgraf, S. Scheuermann, G. Fritz, R. Pipkorn, J.r Reed, K. Beyreuther, T.A. Bayer, G. Multhaup, Alzheimer β-amyloid homodimers facilitate Aβ-fibrillization and the generation of conformational antibodies, J. Biol. Chem. 278 (2003) 35317–35324. [29] C. Zhang, Y. Liu, J. Gilthorpe, J.R.C. van der Maarel, MRP14 (S100A9) protein interacts with Alzheimer beta-amyloid peptide and induces its fibrillization, PLoS ONE 7 (3) (2012) e32953. [30] I.-H. Chou, M. Benford, H.T. Beier, G.L. Cote, Nanofluidic biosensing for β-amyloid detection using surface enhanced Raman spectroscopy, Nano Lett. 8 (6) (2008) 1729–1735. [31] H.S. Shafaat, B.S. Leigh, M.J. Tauber, J.E. Kim, Resonance Raman characterization of a stable tryptophan radical in an azurin mutant, J. Phys. Chem. B 113 (2009) 382–388. [32] A.K. Thakur, C.M. Rao, UV-light exposed prion protein fails to form amyloid fibrils, PLoS ONE 3 (7) (2008) e2688. [33] C.L. Worth, T.L. Blundell, On the evolutionary conservation of hydrogen bonds made by buried polar amino acids: the hidden joists, braces and trusses of protein architecture, BMC Evol. Biol. 10 (2010) 161. [34] T. Kowalewski, D.M. Holtzman, In situ atomic force microscopy study of Alzheimer's β-amyloid peptide on different substrates: new insights into mechanism of β-sheet formation, Proc. Natl. Acad. Sci. U.S.A. Biophys. 96 (1999) 688–3693. [35] M. Lysetska, A. Knoll, D. Boehringer, T. Hey, G. Krauss, G. Krausch, UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study, Nucleic Acids Res. 30 (2002) 2686–2691. [36] M. Arimon, I. D-Perez, M.J. Kogan, N. Durany, E. Giralt, F. Sanz, X. F-Busquets, Fine structure study of Aß1–42 fibrillogenesis with atomic force microscopy, J. Fed. Am. Soc. Exp. Biol. 19 (2005) 1344–1346. [37] Y. Ohko, K. Ichiroiuchi, C. Niwa, T. Tatsuma, T. Nakashima, T. Iguchi, Y. Kubota, A. Fujishima, 17β-Estradiol degradation by TiO2 photocatalysis as a means of reducing estrogenic activity, Environ. Sci. Technol. 36 (2002) 4175–4181. [38] R. Flamia, G. Lanza, A.M. Salvi, J.E. Castle, A.M. Tamburro, Conformational study and hydrogen bonds detection on elastin-related polypeptides using X-ray photoelectron spectroscopy, Biomacromolecules 6 (2005) 1299–1309. [39] M.H. Ahmed, T.E. Keyes, J.A. Byrne, The photocatalytic inactivation effect of Ag–TiO2 on β-amyloid peptide (1–42), J. Photochem. Photobiol. A Chem. 254 (2013) 1–11
