Gold Nanoparticles Can Induce theFormation of Protein-based Aggregatesat Physiological pHDongmao Zhang,†,#,| Oara Neumann,†,# Hui Wang,‡,⊥ Virany M. Yuwono,‡Aoune Barhoumi,‡ Michael Perham,‡ Jeffrey D. Hartgerink,‡Pernilla Wittung-Stafshede,‡,§,+ and Naomi J. Halas*,†,‡

Department of Electrical and Computer Engineering, Department of Chemistry, andDepartment of Biochemistry and Cell Biology, Rice UniVersity, 6100 Main Street,Houston, Texas 77005

Received October 8, 2008; Revised Manuscript Received January 9, 2009

ABSTRACT

Protein-nanoparticle interactions are of central importance in the biomedical applications of nanoparticles, as well as in the growing biosafetyconcerns of nanomaterials. We observe that gold nanoparticles initiate protein aggregation at physiological pH, resulting in the formation ofextended, amorphous protein-nanoparticle assemblies, accompanied by large protein aggregates without embedded nanoparticles. Proteinsat the Au nanoparticle surface are observed to be partially unfolded; these nanoparticle-induced misfolded proteins likely catalyze the observedaggregate formation and growth.

As a virtually inert and highly biocompatible nanomaterial,gold nanoparticles exhibit enormous potential in biotech-nology and biomedicine from protein and DNA detection tocancer therapy and drug delivery.1-4 Recent reports that somenanomaterials catalyze the formation of protein fibrils provideevidence that interactions between proteins and nanophasematerials can induce modifications in protein structure,leading to the growth of extended assemblies. Thus far, thisbehavior has only been observed under extreme conditionsand with materials not in current or imminent in vivo use.Here we report the surprising observation of an interactionbetween certain proteins and Au nanoparticles at physiologi-cal pH that results in the formation of two types of extendedaggregates: protein-nanoparticle assemblies and amorphousprotein aggregate structures. While lysozyme is the proteinwe have studied most extensively, this behavior has also beenobserved to occur with several other proteins, such as avidin,under similar conditions. This behavior is in stark contrastto other well-known protein-Au nanoparticle induced effects,

such as the stabilization of nanoparticles in solution uponadsorption of proteins, which actually inhibits aggregateformation.5 This discovery clearly reflects the complexityof the protein-nanoparticle interaction in light of the diversityof protein structure. Moreover, this effect may also be ofgreat potential importance in the context of nanobiomedicineand may provide a potential mechanism for protein scaveng-ing or other therapeutic applications once more fullyunderstood. Fortuitously, aggregates of Au nanoparticles arewell known to give rise to extremely large local electro-magnetic fields between directly adjacent nanoparticles whenilluminated, known as “hot spots”.6,7 This property allowsfor the detailed study and analysis of the proteins within theseinterparticle spaces using surface enhanced Raman spectros-copy (SERS) at sensitivities that approach the single moleculelevel.8-11 Therefore the observed protein-nanoparticle ag-gregates also provide, by nature of their geometry, a uniqueand unprecedented opportunity to probe a protein-nanopar-ticle interface at the molecular level and obtain specificinformation about protein conformational changes occurringat that interface. This is also a crucially important diagnosticfor this effect, since conventional solution-based proteinanalysis techniques such as circular dichroism (CD) are ofextremely limited use in studying this system due to the rapidremoval of proteins from solution during aggregate forma-tion.

Au nanoparticles (!90 nm in diameter) were fabricatedby reducing 30 mL of the aged chloroauric acid solution (3

* To whom correspondence should be addressed. E-mail: halas@rice.edu.Phone: (+) 1-(713)348-5612. Fax: (+) 1-(713) 348-5686.

† Department of Electrical and Computer Engineering.‡ Department of Chemistry.§ Department of Biochemistry and Cell Biology.# These authors contributed equally to this paper.| Current address: Department of Chemistry, Mississippi State University,

Mississippi State, MS 39762.⊥ Current address: Center for Nano and Molecular Science and Technol-

ogy, The University of Texas at Austin, Austin, TX 78712.+ Current address: Chemistry Department, Umeå University, 901 87

Umeå, Sweden.

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10.1021/nl803054h CCC: $40.75 © 2009 American Chemical SocietyPublished on Web 01/26/2009

mL of 25 mM HAuCl4 with 50 mg K2CO3 dissolved in 200mL of water and aged in dark for 12 h before being used)with 200 µL formaldehyde under rapid stir for 30 min atroom temperature. The resulting colloid solution was cen-trifuged several times and the nanoparticles were redispersedin 20 mL water. The concentration of the gold nanoparticlewas estimated to be 10 pM using absorbance measurementsand Mie scattering theory. All lysozyme solutions wereprepared using 10 mM phosphate buffer (pH ) 7.5). Proteinsolutions were filtered through membranes with 200 nmdiameter pore size to remove possible particulates that maybe present.

Dramatic changes in the extinction spectra of the Aunanoparticles were observed shortly after mixing the nano-particles with a 16 µM lysozyme solution (Figure 1). Thesingle nanoparticle plasmon resonance at !580 nm progres-sively decreases in intensity as a new plasmon band developsin the near-infrared region, arising from the closely spacedinteracting nanoparticles of the aggregate. This change canbe observed even when the lysozyme concentration is as lowas 16 nM (Supporting Information, Figure S1), suggesting asmall dissociation constant (!10 nM or lower) for lysozymeand Au nanoparticles.

A light scattering image, taken in solution, of a representa-tive lysozyme-Au nanoparticle assembly is shown in Figure2A. Confocal light scattering measurement was performedafter overnight incubation of 1.6 µM lysozyme in 5 pM Au-nanoparticle solution. One drop of the lysozyme-Au nano-particle assembly was deposited onto a covered quartz bottomculture dish (MatTek, Inc.) for confocal light scatteringimaging using an inverted Zeiss confocal fluorescencemicroscope. Image shown in Figure 2A was obtained with63" oil immersion objective (NA ) 1.4) with a laserexcitation energy of 543 nm. Three-dimensional assembliesas large as tens of microns are typically observed. This imageclearly shows that Au nanoparticles (appearing as bright spotsin the image) and Au nanoparticle aggregates are distributedthroughout the protein network. More detailed structuralimages of the protein-Au nanoparticle assemblies (usingTEM images) show both isolated Au nanoparticles and

clusters of nanoparticles embedded within a protein matrix(Figure 2B) with no apparent modification of the Aunanoparticle morphology. In addition to these compositeassemblies, large domains of protein aggregates containingessentially no Au nanoparticles were also found. Since it ispossible that the formation of protein aggregates could beinduced by sample (drop-dry) preparation of conventionalTEM images, cryo-TEM images (Figure 2C,D) were per-formed with the frozen solution to unambiguously confirmthat the observed protein aggregates are indeed formed insolution. Some of these protein aggregates have well-definedprofiles (Figure 2D) while others exhibit a random, amor-phous morphology (Supporting Information, Figure S2).TEM and cryogenic TEM experiments and control samplesfor Cryo-TEM measurements were composed of 3.2 µM oflysozyme solution in buffer mixed with equal volume of Aunanoparticle solution and water, respectively. Both solutionswere shaken for !3 h and allowed to sit at room temperaturefor two days before imaging with a JEM FasTEM2010 cryo-TEM Instrument. The samples for TEM experiments wereprepared by drop-drying the solutions on TEM grids. In nocase was protein aggregation found to occur in controlsamples of protein in buffer without the presence ofnanoparticles (Supporting Information, Figure S2). Furtherevidence of protein aggregation in lysozyme-Au nanoparticlesolutions was provided by tryptophan fluorescence measure-ments, which showed that the amount of aggregated lysozymewas far greater than what could be accounted for byadsorption of lysozyme monolayers onto the Au nanoparticles

Figure 1. Extinction spectra of lysozyme-Au nanoparticle mixtureas incubation time increases. Inset is the extinction at 580 nm (blue)and 1000 nm (red) respectively.

Figure 2. Optical and electron microscopy images obtained withlysozyme/Au-NP solutions. (A) Confocal light scattering image ofa lysozyme-Au nanoparticle assembly settled from lysozyme-Aunanoparticle solution. (B) TEM image of dried sample of lysozyme-Au nanoparticle assemblies. (C,D) Cryogenic TEM images ofprotein aggregates, also obtained from lysozyme-Au nanoparticlesolution. It should be noted that some features in images C and Dare of the carbon grid used for the Cryo-TEM measurments.

Nano Lett., Vol. 9, No. 2, 2009 667

present in solution. These observations lead us to concludethat the formation of both the protein-Au nanoparticleassemblies and the protein aggregates without Au nanopar-ticles present are induced by the interaction of proteins withAu nanoparticles.

The protein-Au nanoparticle aggregates are a naturalgeometry for highly enhanced SERS,12 and therefore theirSERS spectra provide a great deal of highly specificinformation about the protein conformation at the protein-Au nanoparticle interface.6,7 In Figure 3, we compare theSERS spectrum of a typical lysozyme-Au nanoparticleassembly (Figure 3A), a normal Raman spectrum of lysozymesolution (Figure 3B), and a Raman spectrum of drop-driedlysozyme on an inert, nonenhancing Teflon substrate (TientaSciences, Inc.) (Figure 3C).13 The SERS spectra obtainedfrom different lysozyme-Au nanoparticle assemblies wereall highly reproducible (Supporting Information, Figure S6),most likely because of densely saturated coverage by theproteins onto the nanoparticle surfaces of the aggregates.Detailed peak assignments of the Raman spectra are listedin Table 1.

The samples for Raman measurements were prepared bydepositing 100 µL of the sample used for cryo-TEMmeasurement onto a spectRIM substrate. Raman spectra wereobtained using a Renishaw InVia Raman microscope, with785 nm excitation laser through a 63" water immersion

objective. The laser power focused on the sample wasmeasured to be 0.57 mW before objective. Solution-phaseand drop-coating-deposition-Raman spectra (DCDR) oflysozyme were acquired with a laser power of 570 mW, andbackground from the buffer solution was subtracted. DCDRspectra were acquired by depositing a high concentration oflysozyme solution onto SpectRIM substrates and wereallowed to dry in a vacuum-assisted desiccator before spectralacquisition by focusing laser beam onto the protein ring.

While the normal, unenhanced Raman spectrum of drop-dried lysozyme (Figure 3B) manifests peptide backbonefeatures very similar to those seen in the solution Ramanspectrum, the SERS spectrum of lysozyme in the protein-Au nanoparticle assembly has several unique features thatindicate conformational modifications for lysozyme in theprotein-Au nanoparticle assembly. For example, the SERSintensity in the !930 cm-1 region, commonly assigned tothe C-C stretch of the R-helical structure, is significantlyreduced in the protein Au-nanoparticle assembly, accompa-nied by a small blue shift of the amide I band from 1655 to1661 cm-1 and a red shift of the amide III band from 1260to 1235 cm-1. These spectral changes strongly suggest thatsome of the R-helical structure of lysozyme may becomemodified to a !-sheet or random coil conformation uponprotein adsorption onto the gold surface.14 Similar confor-mational changes have also been observed for proteinsadsorbed onto macroscopic functionalized gold surfaces.15

It should be pointed out that the SERS spectral modifica-tion in the backbone regions due to the protein/Au nano-particle interaction is far less than one would expect toobserve for protein fibrillation, as fibril formation introducesfar more dramatic changes in peak intensities and spectralshifts in the amide I, III, and C-C regions.16 Therefore thisaggregation appears to be a unique process distinct fromprotein fibrillation, where the lysozyme undergoes only apartial conformational modification on the Au nanoparticlesurface that is not as extensive as the structural changestypical of protein fibrillation.

The Raman features of tryptophan are particularly sensitiveto local environment. The tryptophan signals in the SERS

Figure 3. Raman and SERS spectra of lysozyme measurement at different conditions. (A) SERS spectrum of a representative protein-Aunanoparticle assembly in solution. (B) normal Raman spectrum of lysozyme solution and (C) dried lysozyme deposited on a Teflon substrate.Peak assignment is listed in Table 1, and the dotted lines guide the eye.

Table 1. Peak Assignments for the Raman Spectra Shownin Figure 3

Ramanshift (cm-1) assignment

Ramanshift (cm-1) assignment

296 S-Au21 1103 (C-N) stretch35

504 S-S20 1125 C-N stretch35

524 S-S20 1235 amide III (R helical)36

621 Phe36 1260 amide III (! sheet)36

642 Tyr36 1340 Trp18

760 Trp36 1360 Trp17

855 Tyr36 1421 COO stretch36

877 Trp36 1448 CH bending36

901 C-C36 1535 Trp36

930 C-C(R helical)36

1550 Trp36

1002 Phe36 1583 Trp/Phe36

1012 Trp36 1605 Phe36

1030 Phe36 1661 amide I36

668 Nano Lett., Vol. 9, No. 2, 2009

spectrum are dramatically different than those observed eitherin the solution or in the drop-dried spectra. First, the peakratio of the 1360/1340 cm-1 doublet is greatly increased inthe SERS spectrum, indicating a more hydrophobic sur-rounding for tryptophan residues17 in the protein-Au as-sembly than in the unassociated, native protein. This is likelydue to the exclusion of water at the protein-Au interface andin the ensuing multilayer aggregates in the nanoparticlejunctions. Second, the attenuation of one of the indole ringstretching modes at 1550 cm-1 and the appearance of a newfeature at 1536 cm-1 are indicative of a conformationalheterogeneity of the six tryptophan residues induced byprotein adsorption at the Au nanoparticle surface. Thefrequency of the indole stretching mode has been shown tobe particularly sensitive to the C2-C3-C!-CR dihedralangle, where a frequency of 1550 cm-1 corresponds to adihedral angle of !110° and that of 1535 cm-1 to !60°.18

The observed peak splitting may also indicate that theenvironments of the indole rings of the tryptophan moietiesmay be heterogeneous, also indicating a partial unfoldingof the protein.

Possible covalent interactions between the Au nanoparticleand some subset of the eight cysteine residues in lysozymeare revealed through complex changes in the spectral featuresof the S-S stretch region (500-525 cm-1).19,20 There aretwo likely reasons for these observed changes. First, thereis likely a dissociation of some of the four disulfide bondsas the protein molecule encounters the Au nanoparticlesurface. This correlates with the appearance of a low-wavenumber mode in the vicinity of 300 cm-1 21 character-istic of S-Au bond formation. Second, the dihedral anglein some or all of the four disulfide linkages in the lysozymemolecule may change with conformation; this results inchanges in both peak position and intensity of the S-Sstretching modes.20 When we compare the spectra in Figure3, panels A and C, the complex changes in this spectralregion allow us to infer that some changes in the dihedralangles of the disulfide linkages have occurred, correlatingwith an observed appearance of the Au-S stretch mode at296 cm-1. This latter feature confirms the presence ofcovalent interactions between the Au nanoparticle surfaceand the dissociated disulfide groups of the lysozyme. Thestrong similarity between the SERS spectrum of the protein-Au nanoparticle aggregate (Figure 3A) and the drop driedlysozyme spectrum (Figure 3B) in the S-S stretch regionleads us to conclude that some fraction of the four S-Slinkages per lysozyme molecule are likely to remain intactat the protein-Au nanoparticle surface. Given the importanceof the disulfide bond in maintaining the protein secondarystructure,22 partial breakage of the disulfide bonds alsosupports the notion that the aggregated proteins are partiallyunfolded.

Surprisingly, relative to all other spectral features observed,the ring breathing mode of phenylalanine is by far moresignificantly enhanced in the SERS spectrum of the lysozyme-Au nanoparticle assembly than in the normal Ramanspectrum of the protein solution. Given the critical distancedependence of the SERS signal of the specific functional

group on its distance from the SERS active surface,23 it islikely that one or more of the three phenylanine residues inlysozyme are closely associated with the nanoparticle surface.Previous studies demonstrated that among all the S-S bonds,the disulfide bond formed between cysteine residue 6 and127 is most susceptible to breakage via dithiothreitolreduction,24,25 followed by the one formed by cysteineresidues 76 and 94. Coincidently, the formation of a covalentinteraction of cysteine residue 6 with the Au nanoparticlesurface will necessarily bring the phenylalanine residue 3into close vicinity of that surface, which would directly resultin a large SERS signal enhancement of the phenylalaninemodes. In contrast, the phenylalanine residue closest to eitherof the cysteine residues 76 or 94 is over 30 amino acidsaway in the lysozyme primary structure.26 Therefore, thepreferential enhancement of the phenylalanine residue andthe breakage of only some of the disulfide bonds inferredfrom our SERS measurements strongly suggest that thedisulfide bond cleavage between cysteine residues 6 and 127is the most likely one to occur when lysozyme adsorbs ontothe Au nanoparticle surface. The observation that partialdissociation of disulfide bonds occurs at the protein-Aunanoparticle surface also offers a hypothesis for whyinteractions between some proteins and Au nanoparticles leadto misfolding and aggregation and why some do not.Lysozyme is a smaller (15 kDa) protein with only 4 S-Sbonds; the dissociation of 1 or 2 of these bonds is likely tosignificantly affect protein structure. In contrast, largeproteins such as BSA (67 kDa) with over 17 disulfide bondsare less likely to be significantly perturbed by the dissociationof a few S-S linkages that may occur upon Au nanoparticleadsorption.

From our structural and spectroscopic observations, apathway for the formation of the protein-Au nanoparticleassembly can be proposed (Scheme 1). On the basis of thespectroscopic evidence that there are changes in the confor-mation of lysozyme on the Au nanoparticle surface, we inferthat proteins are partially unfolded upon adsorption onto theAu nanoparticle surfaces (a). The partially unfolded proteinson the Au nanoparticles interact with other proteins insolution, seeding further protein aggregation on the nano-particle surfaces (b), similar to what has been observed onflat Au surfaces where adsorbed proteins serve as a templatefor protein aggregation.27 The protein-coated Au nanopar-ticles coalesce to form protein-Au nanoparticle assemblies(c). The free-standing protein aggregates also observed couldbe formed by two scenarios. In one case, solution proteinsthat have misfolded due to an interaction with the proteinson the aggregate surface but have not been incorporated intothe structure serve as nucleation sites for the formation ofthe aggregates. Another possible scenario may be that smallprotein aggregates desorb from the Au nanoparticle surfacesand subsequently seed further protein aggregation.27 Thissecond mechanism is similar to what has been reported forprotein fibrillation where fibril formation can be seeded withpreformed small protein.28,29

This unexpected formation of protein-Au nanoparticleaggregates, which may be a severe cause for concern when

Nano Lett., Vol. 9, No. 2, 2009 669

bare Au nanoparticles are introduced into physiologicalenvironments, can be entirely suppressed by appropriatechemical functionalization of the nanoparticle surface. Forexample, coating the Au nanoparticle surfaces with thiolatedpoly(ethylene glycol) (PEG), currently in widely accepteduse as a coating material in numerous current and proposedmedical applications, can completely inhibit assembly forma-tion (Figure 4). The effect of PEG functionalization wasexamined by exposing Au nanoparticles to a range of PEGconcentrations, followed by the mixing of the functionalizednanoparticles with lysozyme solution. The UV-vis spectraof these mixtures reveal that when the coating concentrationis sufficiently high (>1 µM in this study), no protein-Aunanoparticle assemblies are observed, even after a three dayincubation period. This observation is analogous with studieson macroscopic flat silicon or Au surfaces which show thatfor PEG surface densities above a certain threshold, proteinadsorption can be completely or significantly inhibited.30,31

The observation that partially PEGylated Au nanoparticlesare still capable of inducing protein aggregation has ex-tremely important implications for biosafety quality controlin biomedical applications; from these experiments it is clearthat the type and extent of surface functionalization of Aunanoparticles may result in very different interactionsbetween the Au nanoparticles and proteins in complexbiological environments.

In conclusion, we have demonstrated that Au nanoparticlescan induce aggregation of certain proteins at physiologicalpH. This has extremely important implications regarding thebiosafety concerns of unfunctionalized or partically func-tionalized Au nanoparticles. The association of proteinmisfolding-aggregation with numerous conditions and dis-eases such as the neurodegenerative diseases, that is,Huntington’s, Alzheimer’s, and Parkinson’s disease, is well

known.32-34 Conversely, these observations also suggest thatAu nanoparticles, when suitably functionalized, combinedwith other materials, or in more complex geometries, maypotentially provide a therapeutic function and may actuallybe developed for the manipulation of misfolded proteins.There may also be potential applications of Au nanoparticlesas scavengers of misfolded proteins, to arrest the progressionof certain disorders while research into their origins andultimate cures advance.

Acknowledgment. This work was supported by the ArmyResearch Office under Grant W911NF-04-01-0203, theRobert A. Welch Foundation Grant C-1222, the NationalScience Foundation Grants EEC-0304097 and ECS-0421108,and Anteon Corporation (D.Z.) under USAF-5408-04-SC-0006.

Supporting Information Available: This material isavailable free of charge via the Internet at http://pubs.acs.org.

Scheme 1. Formation of a Protein-Au NanoparticleAssemblya

a (a) Native globular proteins undergo a conformational change uponadsorption onto the Au nanoparticle, (b) partially unfolded proteins on theAu nanoparticle surfaces seed further protein aggregation, (c) coalescenceof protein-coated Au nanoparticles. Unfolded proteins in solution ordislodged protein aggregates nucleate further aggregation of free-standingprotein aggregate structures.

Figure 4. Comparison of the extinction spectra of lysozyme withAu nanoparticles functionalized with thiolated PEGs over a rangeof differing concentrations. (A) Extinction spectra of lysozyme-PEGylated nanoparticle mixture. Prior to the introduction oflysozyme, the Au NPs were functionalized by mixing equal volumesof 25 pM Au NP solutions with thiolated PEG solutions. (a) 10µM; (b) 1 µM; (c) 100 nM; (d) 10 nM; (e) 1 nM; (f) 10 pM. (B)Extinction coefficient at 580 and 1000 nm as a function of thiolated-PEG concentration.

670 Nano Lett., Vol. 9, No. 2, 2009

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1

SUPPORTING INFORMATION

Lysozyme-Induced Spectral Changes in the Au nanoparticle solution Extinction Spectrum.

Protein solutions of concentrations ranging from 32 µM to 3.2 nM (by factors of 10) were mixed with

equal volumes of Au nanoparticle solutions (~10 pM). The control sample was prepared by the mixing

of Au nanoparticle solution with an equal volume of the buffer used to prepare the lysozyme solutions.

The photograph and extinction spectra shown in Figure S1 were taken after shaking the solution for 3

hours.

Figure S1: Extinction spectra and photograph of Au-NPs mixed with lysozyme of different

concentrations (a: 16 µM; b: 1.6 µM; c: 160 nM; d: 16 nM; e: 1.6 nM; f: 0 nM).

0.6

0.5

0.4

0.3

0.2

0.1

12001000800600400

wavelength (nm)

f

a

b

c

d

e

e d c b a f

2

Au nanoparticle Interaction with BSA. BSA/Au nanoparticle solution was prepared by mixing 20

µM of BSA solution, prepared in 10 mM phosphate buffer solution (pH=7.5) with an equal volume of

~10 pM Au nanoparticle solution. Figure S2 shows the surface plasmon resonance (SPR) spectrum of

the BSA/Au nanoparticle solution measured at different times subsequent to sample preparation. The

spectrum labeled as “0 minutes” was obtained with an equal volume mixture of Au nanoparticle

solution and 10 mM phosphate buffer.

0.6

0.5

0.4

0.3

0.2

0.1

Ex

tin

ctio

n (

Ab

s)

11001000900800700600500

Wavelength (nm)

0 mins

10 mins

35 mins

130 mins

0.56

0.52

0.48

0.44

Ex

tin

ctio

n (

Ab

s)

620600580560540

Wavelength (nm)

Figure S2: SPR spectra of ~5 pM Au nanoparticle in 10 µM BSA solution measured at different times

following sample preparation. Spectra obtained at 10 mins, 35 mins and 130 mins were scaled with a

scaling factor of 1.000±0.003, to compensate for small intensity variations between spectral

acquisitions. The inset shows the spectra in the wavelength region between 530 nm and 620 nm.

Unlike the time-varying extinction spectrum of the Au nanoparticles mixed with lysozyme solution

3

(Fig. S1), there is essentially no temporal variation of the extinction spectra of the BSA/Au nanoparticle

solution. Compared to the spectrum obtained for the BSA-free Au nanoparticle solution, however, the

extinction spectra of Au nanoparticles in BSA is red-shifted 4 nm from 572 nm to 576 nm, indicating

BSA adsorption onto the Au nanoparticles.36, 37 The lack of time-dependent spectral variation in the SPR

spectra of the BSA/Au nanoparticle solution indicates that no Au nanoparticle aggregation occurs after

BSA adsorption: aggregation would necessarily give rise to a new SPR peak at longer wavelength

region (as in Fig. 1 in article). This result is in contrast with the aggregate formation for the

lysozyme/Au nanoparticle solution, where the formation of aggregates is essentially completed in the

first 30 minutes after the mixing of Au nanoparticle solution with lysozyme.

Quantification of Lysozyme Adsorbed onto Au nanoparticles with Tryptophan Fluorescence:

Quantification of lysozyme adsorbed onto Au nanoparticles was performed with highly concentrated

nanoparticles synthesized with the same protocol specified in the experimental section, but with a

doubled amount of formaldehyde. The blue curve in Figure S3 is the extinction spectra of the stock Au

nanoparticle solution after a 60 X dilution, while the red curve is the extinction spectrum calculated

using Mie theory, assuming a particle size of 56 nm diameters. The overlapping of the peak maxima in

the experimental and the calculated spectra indicates an average size of the synthetic Au nanoparticle is

nominally 56 nm. The larger peak width at half maximum of the sample spectrum is due to some

polydispersity of the synthetic Au nanoparticles.

Stock concentration of the synthetic Au nanoparticle was estimated to be 2.8 nM according to its Uv-

Vis spectrum and the extinction coefficient calculated for 56 nm Au nanoparticles.

The red oily stock solution of Au nanoparticles turned into black directly after it was mixed with an

equal volume of 5 µM, 10 µM or 20 µM lysozyme solution respectively. Corresponding controls for

each experiment solution were prepared by mixing the 5 µM, 10 µM or 20 µM lysozyme solutions with

4

equal volumes of distilled water. After shaking both control and experimental solutions for two days

under ambient conditions, all solutions were centrifuged for 30 minutes (400g) with an IEC LC21

multispeed centrifuge machine (Thermo Electron). Fluorescence intensities of tryptophan for each

solution shown in Figure S5 were measured with a fluorolog-3 fluorimeter (Jobin Yvon Horiba Inc.).

The calibration curve shown in Figure S4 (D) was obtained with three control solutions as well as two

lysoyzme calibration solutions with concentrations of 3.75 µM and 6.5 µM, respectively.

Figure S3: Surface plasmon resonance spectra of Au nanoparticle solution. The blue trace was

obtained with theAu nanoparticles after 60X dilution from a stock Au nanoparticle solution, and the red

trace is the calculated SPR spectrum, assuming a particle size diameter of 56 nm. The intensity of the

calculated spectrum is scaled to match the peak maximum of the Au nanoparticles.

5

Figure S4: Fluorescence intensities of tryptophan in the supernatant of lysozyme solutions (red) with

and (blue) without Au nanoparticles. Final lysozyme concentrations are 2.5 µM, 5 µM and 10 µM

respectively. Concentration of Au nanoparticle is 1.4 nM in all the Au nanoparticle containing solutions.

Plot (D) is the calibration curve. The peaks around 310 nm correspond to the Raman modes of H2O.

With the results shown in Figure S4 (C)-(D), it is estimated that ~4000 lysozyme molecules were

centrifuged per Au nanoparticle. Assuming all the precipitated proteins were accumulated onto the Au

nanoparticle surfaces and the adsorbed proteins retain their solution hydrodynamic radius at pH~7,38

there would be over 6 layers of protein on each Au nanoparticle, consistent with our observation of

multilayer protein deposition onto the nanoparticles.

6

Cryo-TEM images taken with the Lysozyme/Au nanoparticle solution and control solution

Figure S5: Cryo-TEM Images of structures obtained from (A)-(C) lysozyme/Au nanoparticle solution

and (D) control solution. While most aggregates in images (A)-(C) appear to be amorphous, some of

them have slightly more defined features, such as relatively straight edges. No aggregates were found in

the control samples. Scale bar: 200 nm.

A B

C D

7

SERS Spectra and Peak Assignment for Protein Adsorbed onto the Au nanoparticle Surfaces

Figure S6: Representative SERS spectra acquired with three different protein/NP assemblies in

solution. All spectra were acquired using a 63X water immersion objective with a laser power of 0.57

mW measured before sample (0.1% of the power used for normal Raman acquisition) and an integration

time of 10 s. Spectra were offset for clarity.

References:

36. Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, (5), 2714-2721. 37. Tam, F.; Moran, C.; Halas, N. J. Phys. Chem. B 2004, 108, (45), 17290-17294. 38. Bonincontro, A.; De Francesco, A.; Onori, G. Colloids and Surfaces B: Biointerfaces 1998, 12,

(1), 1-5.

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