Nanoscale

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State Key Laboratory of Surface Physics an

Shanghai 200433, P. R. China. E-mail: xjya

Cite this: DOI: 10.1039/c3nr02973e

Received 8th June 2013Accepted 12th August 2013

DOI: 10.1039/c3nr02973e

www.rsc.org/nanoscale

This journal is ª The Royal Society of

Influence of Au nanoparticles on the aggregation ofamyloid-b-(25–35) peptides

Qianqian Ma, Guanghong Wei and Xinju Yang*

The influence of Au nanoparticles (Au NPs) on the aggregation of amyloid-b-(25–35) peptides (Ab25–35) is

investigated by atomic force microscopy and Thioflavin T fluorescence measurements. It is found that,

without Au NPs, the Ab25–35 peptides aggregate gradually from monomers and oligomers to long fibrils

with the incubation time. In contrast, short protofibrils are formed quickly after Au NPs are added to

the Ab25–35 solution, which can be further aggregated to form short fibril bundles or even bundle

conjunctions. To reveal the origin of Au NPs on the aggregation of Ab25–35, electrostatic force

microscopy and scanning Kelvin microscopy are employed to investigate the electrical properties of the

Ab25–35 fibrils with and without Au NPs. Due to the significant difference of the electrical properties

between the Ab25–35 fibrils and Au NPs, the locations of Au NPs inside the Ab25–35 fibril bundles can be

revealed and hence a possible influence mechanism of Au NPs on the aggregation of Ab25–35 is suggested.

1. Introduction

The bril formation of amyloidogenic proteins is believed tobe associated with many diseases, such as Parkinson’s, Hun-tington’s and Alzheimer's diseases.1 The brilization processinvolves the transition of a certain amyloid protein fromnormally soluble forms to insoluble amyloid brils, whichaccumulate in the extracellular space of various tissues.2,3

Alzheimer's disease (AD)3 is one of the best-known amyloiddisorder diseases, and it is characterized by the aggregation ofamyloid-b (Ab) proteins in the brain.4 Up to now there are noeffective drugs to cure these diseases and the treatmentoptions are extremely limited, thereby increased attention hasbeen paid to reveal ways to prevent or cure these protein-aggregation diseases.5–9 In recent decades, nanomaterials,especially nanoparticles, have been increasingly applied inbiomedicine for therapeutic and diagnostic purposes. Theutilization of nanoparticles for the treatment of protein-aggregation diseases is an important one among these appli-cations. Because gold is well suited for biological applicationsbecause it is non-toxic and easily detectable, studies on theconjugation of Ab proteins with the surfaces of Au nano-particles (Au NPs) have attracted considerable attention.10–22 Bynow, the phenomena of the inuence of Au NPs on the Abprotein aggregations has already been reported using varioustechniques such as scanning electron microscopy, trans-mission electron microscopy, atom force microscopy andoptical spectroscopies. It has been found that the Au NPs caneither promote or inhibit the aggregation of Ab proteins,

d Physics Department, Fudan University,

ng@fudan.edu.cn

Chemistry 2013

depending on the size and functionality of Au NPs, or the pHvalue of the solution etc.10–22 However, the mechanisticconsequences of Au NPs effects on protein aggregation arestill not completely understood, mainly due to the limitationof the commonly used experimental methods in revealing thedetails of interaction between the nanoparticles and theproteins. In the area of theoretical research, there are alreadya number of molecular simulations focused on the aggrega-tion of Ab proteins, along with a few studies dealing with theinuence of carbon nanotubes on the aggregation of Abproteins.23–26 But researches on the interaction between AuNPs and Ab proteins have not been reported yet, due to thecomputationally huge simulations required to simulate allthe NP atoms, especially for large NPs. The existing moleculardynamic simulations of NPs have been done mostly forsmall Au NP with simple alkanethiol ligands.17,27 Therefore,the studies on the interaction of Au NPs with Ab proteins areworth in-depth investigation, especially with novel experi-mental techniques.

Toaddress theseunresolved issues, here the amyloid-b-(25–35)peptide (Ab25–35) (with sequence GSNKGAIIGLM) was chosen as amodel system and the inuence of Au NPs on the aggregation ofAb25–35 is investigatedbydifferent techniques.Althoughthemajorcomponents of neuritic plaques found in ADare 40- to 42-residue-longAbpeptides (Ab1–40/Ab1–42), shorter fragmentsof peptides arealso concerned, such as Ab25–35. It has been reported that Ab25–35can form readily b-sheet aggregates and is toxic to neurons.28,29

Due to its small size, the pathological aggregation of Ab25–35peptides has been studied extensively by experiments30–32 as wellas computer simulations.33–36However, the inuence of AuNPsonthe aggregation of Ab25–35 peptides has been less investigated,13

thereby further studies on the aggregation of Ab25–35 with Au NPs

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are necessary for the understanding of the interaction betweenAu NPs and Ab25–35.

In this paper, the inuence of Au NPs on the aggregation ofAb25–35 peptides is investigated by atomic force microscopy(AFM) and Thioavin T (ThT) uorescence. Besides thesetraditional measurements, electrical microscopies such aselectrostatic force microscopy (EFM) and scanning Kelvinmicroscopy (SKM) are employed to measure the electricalproperties of Ab25–35 peptides with and without Au NPs. Due tothe large difference in electrical properties between the Ab25–35brils and Au NPs, the locations of Au NPs inside the Ab25–35brils can be revealed, which can assist us to nd out theinuence mechanism of Au NPs on the aggregation of Ab25–35peptides. In previous studies, EFM and SKM have already beenused to investigate the electrical properties of Ab brils.37–40 Forexample, EFM has been applied to distinguish the differenttypes of Ab polypeptide brils (hollow polypeptide tubes andsilver-lled polypeptide tubes).38 SKM was applied to measurethe surface charge distribution of Ab brils and it was foundthat the surface charge distribution of b-lactoglobulin amyloidbrils was critically dependent on protein conformation.39

However these techniques have never been applied to investi-gate the aggregation mechanism of proteins.

2. ExperimentMaterials

Synthetic Ab25–35 was purchased from GL Biochem (Shanghai)Ltd. The peptide was puried by high performance liquidchromatography (HPLC), and the purity was greater than 95%,as identied by mass spectrometer. The peptide solution wasprepared by dissolving 1 mg Ab25–35 peptide in 10 mL deionizedwater (18.2 MU cm�1). To prepare the mixed solutions of Ab25–35with Au NPs, 1 mL freshly prepared peptide solution was mixedwith 10 mL solution of Au NPs which were prepared as below. Allof the solutions were incubated at room temperature withoutstirring.

Synthesis of Au NPs

The solution of Au NPs was prepared by citrate reduction ofHAuCl4.41 Before use, all glass vessels were cleaned by aquaregia for at least 20 min and thoroughly rinsed with freshdeionized water. A 40 mL aqueous solution of 0.01% HAuCl4was stirred mechanically during heating in an oil bath at 130 �C.Aer boiling, a 400 mL of 1% trisodium citrate solution wasadded, and the solution was stirring vigorously for 20 minutes.When the color changed from pale yellow to deep red, thesuspension was cooled to room temperature with stirring at thesame time. The size and monodispersity of the Au NPs werechecked by scanning electron microscopy, giving an averagediameter of 16 nm.

Sample preparations

To prepare the samples for AFM imaging, an aliquot of 10 mL as-prepared or incubated solution was deposited on freshlycleaved mica surfaces. The deposited droplet was le on the

Nanoscale

substrate for 10 s, and then gradually dried under a gentlestream of nitrogen to prevent contamination. For EFM and SKMmeasurements, the samples were prepared by depositing thesolutions on chemically cleaned silicon substrates instead, withthe same produces as above.

SPM and ThT uorescence measurements

The AFM, EFM and SKM measurements are performed oncommercial SPM equipment (Multimode V, Bruker NanoSurface, USA). The topography images of all the samples wereobtained by AFM in tapping mode with Si tips. EFM recordsboth the sample topography and the phase shi by using a two-pass method.37,42 In the lied pass (lied high enough to neglectthe phase shi by van der Waals force), a DC bias of �2 or �3 Vis applied between the tip and the sample, and the phase shisignal determined by the electrical force gradient is measured.For SKMmeasurements, a voltage consisting of a DC bias with asmall AC modulation is applied to the tip. In our measure-ments, an AC bias with an amplitude of 0.3 and 3 V is applied tothe tip when imaging the mixed bundles of Ab25–35 and Au NPsand pure Ab25–35 brils, respectively, at a li height of 20 nm.During the scan, with the help of a feedback system, the DC biasis adjusted to be always equal to the contact potential difference(CPD) between the tip and the sample surface at each point.Thereby CPD distribution of the sample surface can be derivedfrom the applied DC biases.40 Highly n-doped Si tips are appliedin EFM and SKM measurements. All the electrical experimentswere carried out in a owing nitrogen atmosphere at roomtemperature. Typically in each measurement, scanning sizesrange from 2 to 20 mm. The scanning rate is set as 1 Hz forobtaining small scale topography images and 0.5 Hz formeasuring large scale topography as well as EFM and SKMimages, respectively.

The Thioavin T (ThT) dye uorescence measurements wereperformed with F-2500 FL Spectrophotometer (Hitachi) in a1.0 cm quartz cuvette. Samples were prepared by dissolving the200 mL Ab25–35 (100 mM) solution and 50 mL ThT solution (1 mM)in 1 mL deionized water. The control ThT sample was preparedby dissolving 50 mL ThT solution (1 mM) in 1.2 mL deionizedwater. The excitation wavelength of 420 nm was used for theThT uorescence measurements.

3. Results and discussion

The AFM images of pure Ab25–35 aggregates with time are pre-sented in Fig. 1. The aggregation of Ab25–35 with incubation timecan be clearly observed. For the sample deposited with thefreshly prepared solution (Fig. 1(a)), both small and large olig-omers are observed in the topography image. In small scaleimages (not presented here), monomers and dimers can also beobserved. With the incubation time, the monomers and dimerscontinue to aggregate and the oligomers begin to assemble intoprotobrils. In the AFM image taken aer 5 days (Fig. 1(b)), anumber of oligomers together with some protobrils areobserved. From the image taken aer 10 days (Fig. 1(c)), it canbe seen that the amount of protobrils increases while that of

This journal is ª The Royal Society of Chemistry 2013

Fig. 1 AFM images of Ab25–35 as a function of incubation time. (a) Sample wasprepared with the freshly prepared solution; (b), (c) and (d): samples preparedwith the solution incubated after 5, 10 and 30 days, respectively.

Fig. 2 AFM images of Ab25–35 with Au NPs added in the freshly prepared Ab25–35solution as a function of co-incubation time. The topography images are taken at(a) as prepared, (b) 24 hours after, (c) 48 hours after and (d) 120 hours after.

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the oligomers decreases. Signicant aggregation is observedaer 30 days, as shown in Fig. 1(d). A large number of brils aswell as protobrils are formed, together with a small amount ofoligomers. The above results suggest that pure Ab25–35 wouldaggregate from the monomers and oligomers to protobrils orbrils, and the brilization process requires about 30 days.

In the comparative experiments on samples prepared withAb25–35 solution added with Au NPs, the aggregation behaviorsare signicantly different. The topography images of Ab25–35with Au NPs as a function of incubation time are shown inFig. 2. From the image of the sample prepared immediatelyaer Au NPs are added into the freshly prepared Ab25–35 solu-tion (Fig. 2(a)), it can be seen clearly that most of themonomers,dimers and oligomers have been already gathered and only asmall amount of scattered oligomers can be observed. Aer 24hours of co-incubation of Au NPs with Ab25–35 solutions, pro-tobrils, short brils and even short-bril bundles are observed,as shown in Fig. 2(b). Oligomers are no longer observed and theaggregation processes continues with time. The AFM imagestaken aer 48 and 120 hours (Fig. 2(c) and (d) respectively) showthat the short bril bundles are further connected to each otherwhich gradually grow in length but decrease in width. It indi-cates that these bril bundles will be gathered more tightly withtime, most probably to form more ordered secondary struc-tures. From the above results, it can be demonstrated that theaggregation of Ab25–35 with Au NPs is signicantly differentfrom the aggregation of Ab25–35 without Au NPs. As the Au NPsused for this study are citrate-coated, their surfaces are nega-tively charged.43,44 Meanwhile the Ab25–35 fragment contains onepositively charged amino acid and the remainder are neutral. So

This journal is ª The Royal Society of Chemistry 2013

Ab25–35 can be quickly adsorbed on the surfaces of Au NPs dueto the strong electrostatic force between Ab25–35 and Au NPs.This electrostatic force is much larger than the interactionsbetween Ab25–35 peptides, so the aggregation of Ab25–35 with AuNPs should take precedence over the self-aggregation of Ab25–35,which needs further experiments to verify.

Therefore, ThT uorescence of Ab25–35 without and with AuNPs is measured as a function of incubation time in solution tocheck the change of Ab25–35 conformation in absence andpresence of Au NPs respectively, as shown in Fig. 3. The ThTuorescence spectra of the freshly prepared Ab25–35 solutionswith and without Au NPs are presented in Fig. 3(a), togetherwith the control spectrum of free ThT solution. It can be seenthat the uorescence signal of Ab25–35 with Au NPs is larger thanthat of Ab25–35 without Au NPs. As the ThT uorescence inten-sity is proportional to the amount of the b-sheet structures, itcan be indicated that the adding of Au NPs would accelerate theformation of b-sheet structures, even at the initial stage. Theintensities of ThT uorescence of Ab25–35 with and without AuNPs as a function of incubation time are given in Fig. 3(b). Herethe uorescence intensity is deduced by integrating each spec-trum aer subtracting the background uorescence contrib-uted by the free ThT solution, and then divided by the intensityof free ThT to exclude the inuence of laser intensity variationin different measurements. The ThT uorescence intensity ofthe Ab25–35 solution with Au NPs is always larger than thatwithout Au NPs, and both uorescence intensities increase withthe incubation time. So the results indicate that the adding ofAu NPs would promote Ab oligomers to form b-sheet structures.But since Ab protobrils and brils are all b-sheet structures,

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Fig. 3 (a) The ThT fluorescence spectra of freshly prepared Ab and Ab–Au NPssolutions mixed with ThT solution. The spectrum of bare ThT solution is alsomeasured to excluding the influence of laser intensity in different measurements.(b) The fluorescence intensities of ThT bound to Ab25–35 and to Ab25–35–Au NPs asa function of incubation time.

Fig. 4 The topography (a) and EFM (b) images of Ab25–35 deposited on Sisubstrate incubated 12 days after preparation, respectively; (c) height and phaseshift profiles along the marked line in (a). (d and e) The topography and EFMimages of Ab25–35 + Au NPs deposited on Si substrate incubated 10 daysafter preparation, respectively; (f) height and phase shift profiles along themarked line in (d).

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detailed conformational information cannot be obtained fromthe ThT results. On the other hand, the solution of Ab25–35 withAu NPs can change color from reddish at the original stage ofadding Au NPs into to bluish with incubation time. A similarcolor change was also observed by Yokoyama et al., as well as byPuntes et al., when adding Au NPs to Ab1–40/Ab1–42 solutions,and they attributed that color change to the conjunctionbetween Ab1–40/Ab1–42 and Au NPs.10–12,15,16 In our case, we alsoattribute this color change to the conjunction between Ab25–35and Au NPs. By combining the AFM and ThT uorescenceresults as well as the color change, it can be suggested that theadding of Au NPs to a Ab25–35 solution would result in quickconjunctions between Ab25–35 and Au NPs, promoting theformation of large aggregates which are mainly b-sheet struc-tures. However the interaction details between Ab25–35 with AuNPs cannot be revealed from the AFM and ThT uorescencemeasurements.

To understand the inuence mechanism of Au NPs on theaggregation of Ab peptides, it is necessary to reveal the locationsof Au NPs within the Ab brils. Thus EFM and SKM are appliedas the Au NPs and Ab brils have large differences in theirelectrical properties. The electrical phase shi image obtainedby EFM and the surface potential image obtained by SKM onAb25–35 with and without Au NPs are presented in Fig. 4 and 5,respectively. The topography and phase shi images of pureAb25–35 aer 12 days incubation measured at a sample bias of�3 V and a li height of 60 nm are shown in Fig. 4(a) and (b),respectively. The line proles across ve brils along themarked line in Fig. 4(a) are given in Fig. 4(c). It can be seen thatthe ve brils, which are about 5 nm in height, have similar

Nanoscale

phase shis, i.e. 0.1� relative to the Si substrate. Meanwhile, theEFM topography and phase shi images of Ab25–35 with Au NPsaer 10 days incubation are also measured, as shown inFig. 4(d) and (e), respectively. Here a DC bias of �2 V is appliedand the tip is raised 40 nm above the sample. The line prolesalong the bril bundles shown as the marked line in Fig. 4(d)are given in Fig. 4(f). It can be seen that bright dots appeareddiscontinuously inside or intermediate to the bril bundles inthe topography image, which have very large phase shis in theEFM image. From Fig. 4(f), it can be concluded that the brightdots are 20–30 nm in height and have a phase shi of about 1.0�

relative to the Si substrate, much larger than the phase shivalues of the pure Ab brils (�0.1� as shown in Fig. 4(c)). Thephase shis of the lower parts on the bril bundles (consist ofAb peptides) are about 0.1–0.2� relative to the Si substrate,similar to those of pure Ab brils. Thus, our EFM results indi-cated that the Au NPs disperse in the bril bundles discontin-uously, and are connected by peptides.

The topography and corresponding surface potential imagesof pure Ab brils taken at the same area as the EFM image areshown in Fig. 5(a) and (b). Also, the line proles across vebrils along the marked line in Fig. 5(a) are plotted in Fig. 5(c).It can be seen that the ve brils have almost the same CPDvalues, which are about �10 � �15 mV relative to the Sisubstrate. As a comparison, the SKM images of Ab brils withAu NPs are shown in Fig. 5(d) and (e). This is the same area asthe EFM image taken in Fig. 4(d), except that the widths of the

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Fig. 5 The topography (a) and (b) CPD images of Ab25–35 deposited on Sisubstrate after 12 days incubation, respectively, which are taken at the samearea as Fig. 4(a) and (b); (c) height and CPD profiles along the marked line in (a).(d and e) The topography and CPD images of Ab25–35–Au NPs taken at thesame area as Fig. 4(d) and (e); (f) height and CPD line profiles along the markedline in (d).

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brils are widened a little due to the tip-broadening effect. Itcan be still observed, however, that some bright dots appeareddiscontinuously inside the brils in the topography image,which correspond to the dark dots in the SKM image. From theSKM image as well as the line proles (Fig. 5(f)), it can bedetermined that the CPD values of the dark dots are about �0.7� �1.1 V, while the low parts intermediate to these dark dotsare about �0.2 � �0.4 V, relative to the Si substrate. As in SKM,the measured CPD is dened as Ftip � Fsample,45 where Ftip andFsample are work functions of the tip and sample, respectively.The functions of the conductive tips used in SKM are calibratedwith a HOPG sample each time, the work function of which isabout 4.6 eV, as reported in the previous literature.46 Thus thework function of the pure bril is obtained as about 4.62 eV.Aer adding the Au NPs, the work functions are estimated to be4.95–5.2 eV for dark points, which should be Au NPs sur-rounded by brils, close to the work function values of Au NPsreported in the literature which varied from 4.68 eV to 6.24 eV.47

The work functions of the brils connected by Au NPs are about4.78 eV, slightly different from the pure brils, which may beinduced by the local electric elds modied by the presence ofAu NPs.

From the above results, it can be suggested that the presenceof Au NPs in Ab25–35 solution has a signicant inuence onprotein aggregation, as well as their electrical properties. Theappearance of the aggregations of Ab25–35 with Au NPs observedin our experiments have some resemblance to the results of

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Ab1–40/Ab1–42 and lysozyme with Au NPs.10–13,15,16,48 In theprevious literature, the bonding between conjugated Ab and theAu NPs surface was usually thought to be a major factor indetermining the conformation of protein aggregates.10–12 Thisbonding has been assumed to be the electrostatic force betweenthe charged NP surface and proteins,10–13,44 or the hydrophobicinteraction between –CN– or –NH– groups with an anionic goldcolloidal surface,12,22 or the S–Au bond between the peptides andthe Au NPs surface.10,17 An aggregationmechanismwhich can bereferenced to explain our results has been proposed by Zhanget al. to explain the formation of lysozyme–Au NP assemblies,48

based on structural and spectroscopic observations. Theyinferred that proteins were partially unfolded upon adsorptiononto the surfaces of Au NPs, which interact with other proteinsin solution and seed further protein aggregation on the nano-particle surfaces. Another related model was reported by Liaoet al. to explain the effects of negatively charged Au NPs on theaggregation of Ab1–40.22 They suggested that the conjunction orinteraction between Ab1–40 and negatively charged Au NPs couldinhabit amyloid brillation. Though their experimental resultsare somewhat different from ours, our results are consistentwith their model in the initial stages, before the aggregation ofAu NPs. However, because the negative charges on the surfacesof Au NPs would be neutralized by Ab25–35 and the neutralizedAu NPs have a strong tendency to aggregate, short bril bundlesor large aggregates are observed in our case. By adopting theabove viewpoints and considering the specics of Ab25–35 as wellas the EFM and SKM results, an inuence mechanism which issuitable for Ab25–35 is proposed as follows. As Ab25–35 is posi-tively charged and the citrate-coated Au NPs are negativelycharged; when Ab25–35 is adsorbed on the surfaces of Au NPs, aneutralization reaction would occur between positively chargedamino acid side chains and the negatively charged Au NPssurface, resulting in strong conjunctions between Ab25–35 andAu NPs. In addition, the peptides adsorbed on the surface of AuNPs also have the tendency to aggregate with the other peptidesin the solution to form large oligomers or protobrils. On theother hand, since the negative charges on the surface of Au NPsare neutralized, Au NPs have a strong tendency to aggregatetogether.10 Therefore large aggregates (such as short brilbundles or their conjunctions) are formed due to the strongaggregation trends between Au NPs–Ab25–35 aggregates. Thissuggestion is conrmed by our EFM and SKM results, whichexhibit that the Au NPs are packaged in the protein aggregatesdiscontinuously, connected by Ab25–35 peptides.

To assist our expression of the suggested mechanism, asimple diagram for the aggregation with and without Au NPs isdrawn in Fig. 6. When the Au NPs are added to the freshlyprepared Ab solution, monomers and oligomers are quicklyadsorbed on the surface of Au NPs. With incubation time, theoligomers adsorbed on the surface of Au NPs continue toaggregate with the oligomers in solution and develop intoprotobrils or short bril bundles. Then the short brils orbril bundles, including Au NPs, join quickly to form brilbundle conjunctions as the neutral Au NPs have a strong self-aggregation behavior and act as nucleation centers. Therefore,the presence of Au NPs in Ab25–35 solution facilitates oligomers

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Fig. 6 Schematic diagram showing a possible mechanism of Au NPs acting onAb25–35 peptides. Blue clusters indicate structured and unstructured oligomers,and orange spheres indicate Au NPs.

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to be adsorbed on the Au NP surfaces and to form short brils,resulting in the decrease of the amount of oligomers. Theseshort brils are bundled and then joined together by theaggregation of Au NPs, which can also prevent the formation oflong Ab25–35 brils. Therefore the addition of Au NPs, whensuitably functionalized, would result in preferential combina-tion with the Ab peptides, which can prevent the self-aggrega-tion of Ab peptides to some extent. So in our case, no long brilsare observed when Au NPs are added. Therefore it can be sug-gested that the addition of Au NPs may have potential appli-cations in the scavenging of misfolded proteins; this needsfurther research into their origin.

4. Conclusions

In conclusion, the topography changes of Ab25–35 in the absenceand presence of Au NPs are investigated by conventional AFMand ThT uorescence, as well as the electrical microscopies ofEFM and SKM. Besides the electrical properties obtained onsingle brils, the location of Au NPs within the brils can berevealed due to the large electrical difference between the AuNPs and peptides. According to these results, a possible inu-ence mechanism of Au NPs on Ab25–35 brillization is sug-gested. The presence of Au NPs can accelerate the formation ofthe Au NPs–Ab25–35, but inhibit the aggregation between Ab25–35peptides, which may be useful for the control of amyloiddiseases.

Acknowledgements

The authors want to thank Prof. J. Y. Chen for the ThT uo-rescence measurements. This work was supported by theNational Natural Science Foundation of China (no. 11274072,11274075 and 91227102), and Natural Science Foundation ofShanghai (no. 12ZR1401300). We also thank the Seed Fundsupport by the Physics Department of Fudan University.

References

1 E. Zerovnik, Eur. J. Biochem., 2002, 269, 3362–3371.

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2 J. Legleiter, Methods Mol. Biol., 2011, 670, 57–70.3 A. Alzheimer, Allg. Z. Psychiatr. Psych.-Gerichtl. Med., 1907,64, 146–148.

4 J. Hardy and D. J. Selkoe, Science, 2002, 297, 353–356.5 E. de la Fuente, C. Adura, M. J. Kogan and S. Bollo,Electroanalysis, 2012, 24, 938–944.

6 L. Xiao, D. Zhao, W. H. Chan, M. M. F. Choi and H. W. Li,Biomaterials, 2010, 31, 91–98.

7 C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch, S. Lindman,A. M. Minogue, E. Thulin, D. M. Walsh, K. A. Dawson andS. Linse, J. Am. Chem. Soc., 2008, 130, 15437–15443.

8 A. V. Ghule, K. M. Kathir, T. K. S. Kumar, S. H. Tzing,J. Y. Chang, C. Yu and Y. C. Ling, Carbon, 2007, 45, 1586–1589.

9 J. E. Kim and M. Lee, Biochem. Biophys. Res. Commun., 2003,303, 576–579.

10 K. Yokoyama, N. M. Briglio, D. S. Hartati, S. M. W. Tsang,J. E. MacCormac and D. R. Welchons, Nanotechnology,2008, 19, 375101.

11 K. Yokoyama, H. N. Cho, S. P. Cullen, M. Kowalik,N. M. Briglio, H. J. Hoops, Z. Y. Zhao and M. A. Carpenter,Int. J. Mol. Sci., 2009, 10, 2348–2366.

12 K. Yokoyama and D. R. Welchons, Nanotechnology, 2007, 18,105101.

13 R. C. Triulzi, Q. Dai, J. Zou, R. M. Leblanc, Q. Gu,J. Orbulescu and Q. Huo, Colloids Surf., B, 2008, 63, 200–208.

14 M. Mahmoudi, I. Lynch, M. R. Ejtehadi, M. P. Monopoli,F. B. Bombelli and S. Laurent, Chem. Rev., 2011, 111, 5610–5637.

15 M. J. Kogan, N. G. Bastus, R. Amigo, D. Grillo-Bosch,E. Araya, A. Turiel, A. Labarta, E. Giralt and V. F. Puntes,Nano Lett., 2006, 6, 110–115.

16 M. J. Kogan, N. G. Bastus, R. Amigo, D. Grillo-Bosch,E. Araya, A. Turiel, A. Labarta, E. Giralt and V. F. Puntes,Mater. Sci. Eng., C, 2007, 27, 1236–1240.

17 M. E. Aubin-Tam and K. Hamad-Schifferli, Biomed. Mater.,2008, 3, 034001.

18 A. A. Shemetov, I. Nabiev and A. Sukhanova, ACS Nano, 2012,6, 4585–4602.

19 E. M. Smoak, M. P. Dabakis, M. M. Henricus, R. Tamayevand I. A. Banerjee, J. Pept. Sci., 2011, 17, 14–23.

20 X. R. Xia, N. A. Monteiro-Riviere, S. Mathur, X. Song, L. Xiao,S. J. Oldenberg, B. Fadeel and J. E. Riviere, ACS Nano, 2011, 5,9074–9081.

21 S. Laera, G. Ceccone, F. Rossi, D. Gilliland, R. Hussain,G. Siligardi and L. Calzolai, Nano Lett., 2011, 11, 4480–4484.

22 Y. H. Liao, Y. J. Chang, Y. J. Yoshiike, Y. C. Chang andY. R. Chen, Small, 2012, 8, 3631–3639.

23 A. K. Jana, J. C. Jose and N. Sengupta, Phys. Chem. Chem.Phys., 2013, 15, 837–844.

24 A. K. Jana and N. Sengupta, Biophys. J., 2012, 102, 1889–1896.25 H. Y. Li, Y. Luo, P. Derreumaux and G. H. Wei, Biophys. J.,

2011, 101, 2267–2276.26 Z. M. Fu, Y. Luo, P. Derreumaux and G. H. Wei, Biophys. J.,

2009, 97, 1795–1803.27 S. Rapino and F. Zerbetto, Small, 2007, 3, 386–388.

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28 T. Kubo, Y. Kumagae, C. A. Miller and I. Kaneko, J.Neuropathol. Exp. Neurol., 2003, 62, 248–259.

29 Y. G. Kaminsky, M. W. Marlatt, M. A. Smith andE. A. Kosenko, Exp. Neurol., 2010, 221, 26–37.

30 J. H. Ippel, A. Olofsson, J. Schleucher, E. Lundgren andS. S. Wijmenga, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 8648–8653.

31 G. Shanmugam and P. M. Polararapu, Biophys. J., 2004, 87,622–630.

32 M. Naldi, J. Fiori, M. Pistolozzi, A. F. Drake, C. Bertucci,R. L. Wu, K. Mlynarczyk, S. Filipek, A. De Simone andV. Andrisano, ACS Chem. Neurosci., 2012, 3, 952–962.

33 G. H. Wei and J. E. Shea, Biophys. J., 2006, 91, 1638–1647.34 G. H. Wei, A. I. Jewett and J. E. Shea, Phys. Chem. Chem. Phys.,

2010, 12, 3622–3629.35 M. Kittner and V. Knecht, J. Phys. Chem. B, 2010, 114, 15288–

15295.36 L. Larini and J. E. Shea, Biophys. J., 2012, 103, 576–586.37 C. H. Clausen, M. Dimaki, S. P. Panagos, E. Kasotakis,

A. Mitraki, W. E. Svendsen and J. Castillo-Leon, Scanning,2011, 33, 201–207.

38 C. H. Clausen, J. Jensen, J. Castillo, M. Dimaki andW. E. Svendsen, Nano Lett., 2008, 8, 4066–4069.

This journal is ª The Royal Society of Chemistry 2013

39 G. Lee, W. Lee, H. Lee, S. W. Lee, D. S. Yoon, K. Eom andT. Kwon, Appl. Phys. Lett., 2012, 101, 043703.

40 P. Zhang and H. F. Cantiello, Appl. Phys. Lett., 2009, 95,033701.

41 J. Turkevich, P. C. Stevenson and J. Hillier, Discuss. FaradaySoc., 1951, 11, 55–75.

42 X. H. Qiu, G. C. Qi, Y. L. Yang and C. Wang, J. Solid StateChem., 2008, 181, 1670–1677.

43 J. Turkevich, G. Garton and P. C. Stevenson, J. Colloid Sci.,1954, 9, 26–35.

44 S. H. Brewer, W. R. Glomm, M. C. Johnson, M. K. Knag andS. Franzen, Langmuir, 2005, 21, 9303–9307.

45 A. Avila and B. Bhushan, Crit. Rev. Solid State Mater. Sci.,2010, 35, 38–51.

46 M. M. Beerbom, B. Lagel, A. J. Cascio, B. V. Doran andR. Schlaf, J. Electron Spectrosc. Relat. Phenom., 2006, 152,12–17.

47 W. M. H. Sachtler, G. J. H. Dorgelo and A. A. Holscher, Surf.Sci., 1966, 5, 221–229.

48 D. Zhang, O. Neumann, H. Wang, V. M. Yuwono,A. Barhoumi, M. Perham, J. D. Hartgerink, P. Wittung-Stafshede and N. J. Halas, Nano Lett., 2009, 9,666–671.

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