Article pubs.acs.org/JPCB
Biofunctionalization of Plasmonic Nanoparticles with Short Peptides Monitored by SERS Emma Jorgenson and Anatoli Ianoul* Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada S Supporting Information *
ABSTRACT: In order for plasmonic nanoparticles to be usable in biomedical applications their surface requires functionalization with biocompatible material. For this purpose short peptides, CFY, CFFY, CLY, were designed and replacement of the capping agent poly(vinylpyrrolidone) (PVP) on the surface of silver nanocubes by the peptides was investigated. The primary sequences of the peptides were designed such that they enable the covalent attachment to silver via the cysteine thiols, contain amino acids that can interact via hydrophobic interactions, and therefore are likely to form tightly packed films. Finally, the peptides contained UV−vis and SERS markers, allowing the dynamics of the biomolecule attachment to the nanoparticles to be monitored spectroscopically. The ligand exchange was observed for nanocubes suspended in solution and supported on a dielectric substrate. Formation of the peptide film around the nanocubes was confirmed by electron microscopy and SERS measurements. The film thickness was found to be 4−6 nm and independent of peptide solution concentration, suggesting multilayer formation. The surface density of these cysteine-containing peptides was found to be between 0.59 and 4.92 molecules per nm2.
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INTRODUCTION Applications of plasmonic nanoparticles as intracellular markers often involve their use in environments that can be sensitive or damaging to the particles, which makes surface modification necessary for their efficient usage. The molecules attached to the surface determine how the particles interact with their environment and each other. Capping agents used during the synthesis of nanoparticles, especially for shape control,1,2 may not be ideal for intended application of such nanoparticles. As a result, ligand exchange is often required as it gives the nanoparticles new properties. For example, hydrophobic capping agent, oleic acid, was replaced with silanes with a variety of attached hydrophilic functional groups.3 After ligand exchange (carboxylic acid, amino, and PEG silanes) the particles could be suspended in aqueous solution without aggregation, whereas initially the particles were suspended in hexanes.3 Surface modification with ammonium terminated PEG has been shown to increase nanoparticle binding to DNA compared to a control.4 Similarly, replacing the capping agent on nanoparticles with biomolecules, such as peptides, extends the possible applications of the nanoparticles, allowing their use in drug delivery,5 catalysis,6 the treatment of skin infections7 and healing wounds,8 inhibition of fibrillation and cytotoxicity,9 altering enzyme activity,10 and colorimetric detection of matrix metalloproteinases.11 As such an understanding of peptide characteristics that facilitate monolayer formation on nanoparticles is necessary for these applications to be realized. In several previous studies modifications of various types of nanoparticles, such as gold,12 silver,13 metal oxide,14 and silica15 with peptides have been © XXXX American Chemical Society
investigated. Formation of a monolayer of peptides and other molecules on a surface involves an “anchor” functional group that can attach to the surface. Sulfur containing molecules (amino acid cysteine) can strongly bond to the surface of silver and gold.12 Similarly, other functional groups, including amino groups, can interact with these surfaces by physisorbing.12 CALNN, a cysteine-containing pentapeptide, has been widely studied in terms of monolayer formation on the surface of nanoparticles.12,16 A variety of short peptides based on the sequence CALNN were investigated and demonstrated the importance of a functional group that can bond strongly with the surface, as well as attractive interactions between neighboring strands, to facilitate tight packing of the peptides.12 While strong intermolecular interactions are desirable for increasing the density and therefore robustness of monolayers on the nanoparticle surface, they can often lead to peptide aggregation and formation of complex multimeric structures, for example fibrils. Fibrils have been well studied, in relation to many diseases.17 However, many other peptides can form fibers.18 Self-assembly has been demonstrated for peptides as short as dipeptides, which can form a variety of structures, including fibrils, tubes, vesicles, films, and gels; in addition, selfassembly has been demonstrated for amino acids with modified termini.18 Isomeric tetrapeptides containing aspartic acid and phenylalanine have been shown to form fibrils.19 Many short peptide sequences that are known to produce fibrils, including NFGAI, FGAIL, NFLVH, DFNK, GAIL, GFIL, KLVFF, Received: November 21, 2016 Revised: January 13, 2017 Published: January 13, 2017 A
DOI: 10.1021/acs.jpcb.6b11708 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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150 °C. Next, 25 μL of 0.48 M NaCl was added to the flask, followed by 175 μL of 0.003 M Na2S. Finally, 1.5 mL of 0.28 M AgNO3 was injected at a rate of 0.5 mL/min. The reaction was monitored by UV−vis spectroscopy, and upon the appearance of a peak at 350 nm a second injection of 1.5 mL of 0.28 M AgNO3 at 0.5 mL/min was added. The reaction was quenched in an ice bath when the UV−vis spectra had a dipole peak at 480 nm and an additional sharp peak at 350 nm. Modification of Silver Nanocubes with Peptides. Silver nanocubes (0.16 mL of ∼1.3 × 1014 np/mL) were added to 1.5 mL of three initial concentrations (1.5 mM CFY, CLY, CFFY, and 0.71 mM SFY; 3 mM CFY, CLY, CFFY, SFY; 8.5 mM CFY, CFFY, SFY, and 8 mM for CLY) of peptide solution for 24 h, using 95% ethanol as the solvent. After 24 h the solutions were centrifuged at 12 000 rpm for 10 min and the supernatant was removed and replaced with fresh ethanol, a minimum of four times. UV−vis spectra were taken of the supernatants, and the decrease in concentration from the initial concentration was used to estimate the amount of peptide attached to the surface of the particles. Monolayers of Silver Nanocubes by Langmuir− Blodgett Method. A Nima 311D Langmuir−Blodgett trough was filled with Milli Q water (18.2 MΩ cm) to prepare the glass slides with silver nanocubes. A sample of the particles was resuspended in chloroform and dispersed at the air−water interface by a micropipette. After allowing the chloroform to evaporate the particles were deposited onto the substrates by vertical dipping by removing the slides at 2 mm/min. Modification of Silver Nanocubes Supported on Glass Slides with Peptides. The LB monolayers of PVP-capped silver nanocubes were submerged in solutions of 1.5 mM peptide. These monolayers were removed after incubation times of 5 min, 30 min, and 2.5 h; rinsed thoroughly with ethanol; and characterized with SERS. Surface Enhanced Raman Spectroscopy. The excitation was done with an Ar/Kr laser (Coherent) at 514 nm through an inverted microscope (Olympus IX-71) with a 20× objective. Raman spectra were collected with a single grating monochromator (Jobin Yvon, 640 mm focal length, 1200 lines/mm) with a CCD camera (Princeton Instruments) cooled with liquid N2. Cyclohexane was used for the Raman shift calibration. The data was collected with WinSpec/32 software and processed in GRAMS/AI spectral data processing software. Topographical Measurements. The topography of the NC monolayers transferred onto solid substrates was obtained using an Ntegra (NT-MDT, Russia) atomic force microscope in semicontact mode in air at 21 °C with 512 × 512 points per image. A 100 × 100 μm2 scanner (Ntegra) and cantilevers with rotated monolithic silicon tips (135 μm long, 0.3−6 N/m spring constant NSG03, resonance frequency 90 kHz, NTMDT) were used for all topographic measurements. The typical scan rate was 0.5 Hz. Atomic force microscopy (AFM) images were further processed by Nova image processing software.
DFNKF, NFGSV, Boc-AUV-Ome, Boc-AUI-OMe, FΔF, CbzFF, IF, Fmoc-F, Fmoc-FF.18 Complex multimeric structures that these peptides form can be useful for tissue engineering and drug delivery.18 Plasmonic nanoparticles can interact with these types of peptides, to produce complex novel nanoarchitectures, including chains of nanoparticles assembled onto peptide fibers.17 Hydrogels have been previously prepared using a tripeptide sequence (Boc-Phe-Phe-Ala-OMe), which acts as a gelator, through formation of fibrils. The hydrogels were prepared in combination with silver nanoparticles capped with Cys, Cys-Phe, and Cys-Leu, which can alter the mechanical strength of the gels.20 In addition, the mechanical strength of the resulting gels were also affected by the molecule capping the particles,20 indicating the importance of the molecule on the surface. Hence, understanding characteristics of peptides that allow monolayer formation and replacement of the capping agent on nanoparticles is necessary for the particles to be used in many applications, such intracellular optical labeling or fabrication of complex plasmonic nanoarchitectures with new properties. In this work three peptide sequences, cysteine-phenylalaninetyrosine (CFY), cysteine-phenylalanine-phenylalanine-tyrosine (CFFY), and cysteine-leucine-tyrosine (CLY), were selected to investigate the replacement of PVP on silver nanocubes with peptides. A control peptide serine-phenylalanine-tyrosine (SFY) was also prepared and used in the study. Cubic nanoparticles are of interest due to their unique plasmonic properties, such as ability to support higher order plasmon modes and strong Raman signal enhancement seen at sharp corners and edges.21−24 At the same time, a significant portion of a nanocube surface is flat. This could influence the peptide first layer surface quality (density, stability) and therefore nanocube physicochemical properties, as compared to spherical nanoparticles. The as synthesized nanocubes are functionalized with poly(vinylpyrrolidone) (PVP) which acts as a surfactant but also as a shape controlling agent.1,2 The polymer forms a strong bond with the metal surface through charge transfer interactions.25−27 Thiol containing molecules are however known to replace the polymer.25 Peptides with a thiol containing amino acid cysteine are compared to a peptide with serine in the same position. Serine was selected as it has the same structure as cysteine except for having an oxygen atom, which does not covalently bond with silver, in place of a sulfur atom. As such, the serine containing peptide (SFY) would only be able to attach to the surface through physisorption. In addition, the lengths of the peptides are varied between three and four amino acids. Longer peptides have previously been shown to be more resistant to induced aggregation,12 which is necessary for their efficient usage. The ends were modified for all peptides, C-terminus with ester (OMe) and N-terminus with acetylation (Supporting Information S1).
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EXPERIMENTAL SECTION Materials. Silver nitrate (AgNO3, 99+%), poly(vinylpyrrolidone) (PVP, MW ∼ 55 000), anhydrous 99.8% ethylene glycol (EG), sodium sulfide, and sodium chloride were purchased from Sigma-Aldrich. All peptides were purchased from GenScript (95%). Termini were modified for all peptides, C-terminus with ester (OMe) and N-terminus with acetylation. Synthesis of Silver Nanocubes. Silver nanocubes were synthesized by the polyol process.1,2 Specifically, 35 mL of ethylene glycol (EG) was heated with 0.4 g of PVP for 1 h at
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RESULTS AND DISCUSSION Selection of Peptides for Surface Functionalization. Several factors were considered when designing the peptides for surface functionalization of silver nanocubes. The first was the presence of a thiol containing amino acid (cysteine) in the first position. Thiols are known to strongly attach to the surface of silver nanoparticles, and have been demonstrated to replace PVP on the surface.25 As such, the thiol containing peptides are B
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The Journal of Physical Chemistry B expected to replace PVP on the silver nanocubes’ surface. To confirm the specificity of binding a test peptide was prepared with the same primary structure except for the cysteine replaced with serine. The second factor considered was the intermolecular interactions between peptide molecules. Attractive interactions between amino acids in the middle positions of neighboring molecules, such as hydrophobic interactions and hydrogen bonding, can facilitate the formation of a tightly packed monolayer on the surface of nanoparticles.12 For this work phenylalanine and leucine were chosen for the middle position of the peptide strands, as both are nonpolar amino acids with comparable hydrophobicities.28,29 All peptides contain amino acid tyrosine in the final position of the sequence. Phenylalanine and tyrosine were selected as they are surface enhanced Raman scattering (SERS) and UV−vis spectroscopy markers, which allows the attachment of the peptide to the surface of the particle to be confirmed. The length of the peptide is another crucial parameter we looked to probe, as plasmonic effects decay exponentially with the distance from nanoparticle. As such shorter peptides are preferred. At the same time, monolayers formed from longer peptides are expected to be more robust.12 Therefore, in this work we used peptides that are three and four amino acids in length. Finally, both the C- and N-termini of the peptides were modified to minimize the pH dependence. Specifically, the Nterminus was modified with acetylation and the C-terminus was modified with ester (OMe) (Supporting Information S1). After these considerations, the following peptides were chosen: CFY, CLY, and CFFY. In order to test the specificity of thiol binding, a control peptide SFY was compared with CFY. Assuming a fully extended conformation for the peptides, their lengths should be in the 1 to 1.5 nm range, which for a perfect monolayer provides sufficiently close distance to the surface of nanoparticle to benefit from the plasmonic effects. However, as we show in the later section, the peptides formed multilayers, rather than monolayers. Surface Modification of Silver Nanocubes in Solution. To exchange the PVP ligand with desired peptide suspensions of silver, nanocubes were added to peptide solutions of variable concentrations (initially, 1.5 mM (0.71 mM for SFY), 3 mM, and 8.5 mM (8 mM for CLY)) and incubated for 24 h. The particles were then washed by centrifugation and the supernatant was replaced with fresh ethanol a minimum of four times. The supernatant was collected and used to determine the amount of peptide left on the surface of the particles. Specifically, the concentration of peptide in the first supernatant was determined, based on the intensity of the peak at 277 nm, corresponding to the aromatic side chain,30,31 with the use of a calibration curve (Supporting Information S2) prepared for each peptide. The difference in concentration between the initial solution and the supernatant was estimated to be the amount of peptide on particles’ surface (Table 1). It should be noted that polyvinylpyrrolidone (PVP) also absorbs in the UV region at 215 nm (Figure 1), which made it necessary to use the peak at 277 nm (aromatic side chain)30,31 and not the one at shorter wavelength. Using this approach we determined that for the cysteinecontaining peptides the estimated surface density of peptide on the nanoparticles remains fairly constant with increasing the initial peptide concentration, signaling saturation of the surface sites. The surface density for these peptides ranged from (0.59
Table 1. Estimated Surface Density of Peptides on Silver Nanocubes based on Supernatant UV−vis surface density/molecules/nm2 initial peptide (mM) 1.5 (0.71) 3 8.5 (8) average
CFY 1.32 0.59 0.83 0.91
± ± ± ±
0.76 0.69 0.07 0.37
CFFY 1.88 1.52 1.62 1.67
± ± ± ±
0.16 0.34 0.95 0.18
CLY
SFY
± ± ± ±
0.07 ± 0.02 0.32 ± 0.07 1.85 ± 0.66 -
1.33 4.92 2.25 2.84
0.36 0.62 0.67 1.86
Figure 1. UV−vis spectra of polyvinylpyrrolidone and peptides CFFY, CFY, CLY, and SFY.
± 0.69) nm−2 (for 3 mM CFY) to (4.92 ± 0.62) nm−2 (for 3 mM CLY) (Table 1). However, it is observed that the average surface densities of the cysteine-containing peptides is greatest for CLY (2.84 ± 1.86) nm−2, followed by CFFY (1.67 ± 0.18) nm−2, and lowest for CFY (0.91 ± 0.37) nm−2, indicating that CLY packs the tightest out of these peptides. At the same time, a significant increase in the apparent amount of peptide per unit surface area was seen for SFY with increasing initial peptide concentration, and ranged from (0.07 ± 0.02) nm−2 to (1.85 ± 0.66) nm−2 for initial concentrations 0.71 to 8.5 mM. Such concentration dependence clearly indicates that SFY has not reached saturation at these concentrations, as was observed for the cysteine-containing peptides. To probe the change in the physicochemical properties of the peptide functionalized nanocubes their UV−vis spectra were collected before and after the exchange reaction (Figure 2). The spectra of initial PVP coated nanocubes (Figure 2, black lines) display characteristic peaks with maxima around 350, 400, and 480 nm, corresponding to various localized surface plasmon resonances (LSPR).32,33 After functionalization with cysteine containing peptides (CFY, CFFY, CLY) nanocubes display broadening of the LSPR spectra (Figure 2B−D), indicating some aggregation. This was also observed as darkening of the solution and the particles sedimenting out of solution more rapidly. At the same time the nanocubes incubated in SFY behaved the same as the PVP-capped nanocubes. The UV−vis spectra of the particles modified with SFY did not show broadening (Figure 2A), and the solution did not darken, or sediment out of solution more rapidly than the initial cube sample. In addition the nanoparticles’ incubated in cysteine-containing peptides (Figure 2B−D) show a shift in the LSPR band, which is not observed after incubation in SFY (Figure 2A). After incubation in each peptide the intensity of C
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Figure 2. Sample UV−vis spectra of silver nanocubes before (black) and after incubation in peptide (red), for (A) SFY, (B) CLY, (C) CFY, (D) CFFY (lowest peptide concentration).
the LSPR band drops, which suggests that some nanoparticles were lost during the cleaning stages, however the decrease in intensity is much greater for the cysteine-containing peptides, which could be in part from aggregation as well. Transmission electron micrographs (TEM) were further obtained for the silver nanocubes before and after modification with the peptides (Figure 3). A film is observed around the particles after incubation in 3 mM CFFY (Figure 3F), which is not present before (Figure 3C). A web of peptide is present around the particles after incubation in 3 mM CFY (Figure 3E) (Supporting Information S3). It can be observed that some of the nanocubes experienced rounding of the corners and edges after incubation in 3 mM CLY (Figure 3D). Some peptide can also be observed around these particles as well (Figure 3D). These results indicate that the cysteine-containing peptides are able to attach to the surface of the silver nanocubes. With the use of ImageJ data processing software the film thicknesses were measured from the TEM images. The average film thicknesses were (6.4 ± 2.3) nm for CFY, (5.6 ± 1.8) nm for CFFY and (4.3 ± 2.5) nm for CLY. Interestingly, CFY formed fibril-like structures that were attached to the cube surface (Supporting Information, S3). Similarly, SFY formed fibrils, however these were not attached to the cube surface and were instead observed in the supernatant, which was dropcast and AFMs were obtained (Supporting Information, S4). As is demonstrated with both peptides CFY and SFY, there is a
Figure 3. Silver nanocubes before and after 24 h incubation in peptide solutions and centrifugation to remove excess peptide: (A) reference silver nanocubes before incubation, (B) silver nanocubes after incubation in 0.71 mM SFY, (C) silver nanocubes before incubation, (D) silver nanocubes after incubation in 3 mM CLY, (E) silver nanocubes after incubation in 3 mM in CFY(F) silver nanocubes after incubation in 3 mM CFFY.
potential for these peptides to assemble into larger aggregates. However, as indicated with the TEM images, the presence of the thiol is necessary for these aggregates to attach to the nanoparticle’s surface (Figure 3E). D
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excitation profile changes as well. This however should not influence our conclusions on the completion of the ligand exchange process. SERS spectrum of the initial sample of PVP capped silver nanocubes in suspension (Figure 4, gray) contains peaks at 852, 890, 927, 1061, 1097, 1129, 1295, 1400, and 1664 cm−1. After incubation in CFY (Figure 4, blue) and CFFY (Figure 4, pink) new peaks appear in the spectra at 1001 and 1032 cm−1 (F, ring deformation), 1210 cm−1 (out of plane CH bend/ring deformation).28 After incubation in CLY (Figure 4, orange) there are peaks at 830 (tyrosine, in plane ring breathing) and 850 cm−1 (out of plane ring bend).28 In addition, peaks associated with PVP disappear, indicating successful replacement of the polymer with peptides. Note that the SERS spectra of the nanocubes after incubation in the cysteine-containing peptides, are of the nanoparticles incubated in the lowest peptide concentrations, indicating that theses peptides successfully replace PVP even at these concentrations. After incubation in the highest SFY concentration (Figure 4, green) it is observed that the peak at 1664 cm−1 disappears and a peak at 1001 cm−1 (F, ring deformation)28 has started to develop, however all other peaks were still present in the spectra. These results indicate that SFY is not able to replace PVP on the nanoparticle’s surface, even at the highest experimental concentration, however some peptide seems to be able to attach. These results indicate the importance of the thiol group for the peptide to successfully replace PVP. Surface Modification of Silver Nanocubes Supported by Glass Slides. An initial trial using peptide CFY was conducted by incubating slides in 0.1 mM CFY (Supporting Information, S7). It was observed that peptide was attaching to the surface, as indicated by development of peaks at 1032 cm−1 (F, ring deformation)28 and 1210 cm−1 (out of plane CH bend/ring deformation).28 However, at this concentration the peptide was not able to replace the PVP on the surface, as can be seen by the presence of PVP’s carbonyl (1770 cm−1) in all spectra. These results indicated that the concentration or time frame needed to be increased in order to facilitate the replacement of PVP by peptide. The concentration of peptide was increased to 1.5 mM and the kinetics of ligand exchange on monolayers of silver nanocubes on glass slides was assessed. Silver nanocube monolayers were prepared by the Langmuir−Blodgett method and incubated in 1.5 mM solutions of each of the peptides for various times. Specifically, the slides were removed from the solutions at 5 min, 30 min, and 2.5 h time intervals and rinsed thoroughly with ethanol, to remove any nonspecifically bound peptide. SERS spectra were measured for these samples, as well as a reference (a monolayer of silver nanocubes that had not been incubated in peptide). The carbonyl peak is easier to observe in the cubes supported on glass slides (Figure 5), compared to in solution (Figure 4 gray), making the exchange more easily monitored for the supported nanocubes. The initial spectra (measured before incubation with the peptide) have several peaks associated with PVP on silver nanocube monolayers33 (Figure 5A, C, E, G). Specifically, 1001 cm−1 (C−C and CH2 rock), 1410 cm−1, 1425 cm−1 (CH2 scissor), 1600 cm−1, and 1765−1770 cm−1 (CO) are present in the spectra. With increasing incubation time, peaks associated with PVP decreased, while peaks due to the peptide increased for the cysteine-containing peptides. For the nanocubes incubated in CFY (Figure 5E,F) and CFFY (Figure 5G,H) intensity of the peak at 1001 cm−1
In addition, TEM images were obtained for nanoparticles incubated in other concentrations of CFFY (Supporting Information, S5). These images show consistent film thickness over different initial concentrations (1.5, 3, 8.5 mM) as expected from the estimated constant surface density (Table 1) of (1.67 ± 0.18) nm−2 on average. Based on DFT studies of cysteine on Ag (111) the monolayer density is expected to be in the range of 0.88 nm−2 to 4.56 nm−2.34 At the same time for tripeptides bound to the gold surface via a 3-mercaptopropionic acid linker the surface densities were found to be in the 1.3 to 3.1 molecules per nm2 range.35 All of the estimated surface densities in the present work were under this highest monolayer value except for 3 mM CLY (Table 1). However, the expected lengths of the peptides were in the range of 1−1.5 nm, which suggests that the peptides might not have been packing as tightly under these conditions, as the measured film thicknesses were greater than of a monolayer (Figure 3). Energy dispersive X-ray (EDX) spectra were obtained for the cubes that had been incubated in cysteine containing peptides. The EDX spectra show the presence of sulfur, which is not detected in the EDX spectrum of a reference cube sample (Supporting Information S6). This is consistent with the replacement of PVP with the peptides on the surface for cubes incubated in CFY, CFFY, and CLY. TEM revealed no difference in nanoparticle morphology after incubation in 0.71 mM SFY (Figure 3B), however fibrils were observed in dropcasts of the supernatants (Supporting Information S4). Moreover, no peptide aggregates/multimers were seen attached to the nanocubes, and UV−vis data did not suggest aggregation had occurred (Figure 2A) after incubation in SFY. These results suggest that SFY was unable to replace PVP on the nanocube’s surface, unlike the cysteine-containing peptides. Finally, the presence of the peptide on the surface of the particles after the exchange was confirmed with SERS (Figure 4). Since UV−vis spectra of nanocube slightly change after the ligand exchange reaction (Figure 2) it is possible that the SERS
Figure 4. Surface enhanced Raman spectra of the silver nanocubes in solution initially and after incubation in peptides (lowest concentration for cysteine-containing, highest concentration for SFY). SERS measurements were done on several points and the spectra were averaged, displayed is the average and standard deviation for each spectrum. E
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Figure 5. SERS of silver nanocubes supported on glass slides incubated in 1.5 mM peptide solutions for 0 min (gray), 5 min (green), 30 min (blue), and 2.5 h (pink), for peptides (A) SFY, (C) CLY, (E) CFY, and (G) CFFY. Average peak heights over time black (1770 cm−1, carbonyl) and (B) SFY, red 1001; (D) CLY, red 1001; (F) CFY, red 1032; and (H) CFFY, red 1032. SERS measurements were done on several points and the spectra were averaged, displayed is the average and standard deviation for each spectrum.
(phenylalanine, ring deformation)28 increased overall, while of the peak at 1770 cm−1 (PVP’s carbonyl)33 decreased over time. However, to minimize the interference from the PVP related peak at 1001 cm−1 the bands at 1032 cm−1 (F, ring
deformation)28 were used to monitor the peptide attachment to nanocubes for CFY and CFFY, while a decrease in PVP’s carbonyl (1770 cm−1) allows the removal of PVP from the surface to be monitored. F
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The Journal of Physical Chemistry B Both peaks at 1032 cm−1 (F, ring deformation)28 and 1210 cm−1 (out of plane CH bend/ring deformation)28 increase with increasing incubation time, indicating that peptide is attaching to the surface (Figure 5E,G). These results indicate that CFY and CFFY were replacing PVP on the silver nanocube’s surface. In addition, after incubation for 2.5 h several peaks are observed in the spectra that are associated with the peptide. For both slides incubated in CFY (Figure 5E) and CFFY (Figure 5G) for 2.5 h the SERS spectra show peaks at 1001 and 1032 cm−1 (F, ring deformation), 1210 (out of plane CH bend/ring deformation), and 1607 cm−1 (F, CO stretch).28 The slides incubated in CFY for 2.5 h (Figure 5E) have additional peaks, at 830 (tyrosine (Y), in plane ring breathing), and 850 cm−1 (Y, out of plane ring bend),28 which are also associated with the peptide. For the slides incubated in CLY it was observed that the peak at 1770 cm−1 decreased over time (Figure 5C,D), indicating the replacement of PVP by the peptide (CLY does not contain phenylalanine, and so the decrease in peak height at 1001 cm−1 is also consistent with the replacement of PVP by the peptide). In addition to the loss of peaks associated with PVP, peaks associated with CLY develop over time, specifically, peaks at 830 (Y, in plane ring breathing) and 850 cm−1 (Y, out of plane ring bend).28 These results indicate that PVP is being replaced by CLY on the silver nanocube’s surface. For the slides incubated in SFY there was an overall decrease in the peak height of both 1001 and 1770 cm−1 (Figure 5A,B). In addition, no other peaks that are associated with peptide attachment are observed; specifically, the peaks at 1032 and 1210 cm−1, which were used to monitor the attachment of CFY and CFFY, are not observed to develop. These results do not support the replacement of PVP by SFY, they suggest that although some PVP might be removed from the surface during the rinsing steps, peptide is not attaching. From the peak heights (Figure 5B, D, F, H) it can be noted that the decrease in PVP’s carbonyl (1770 cm−1) decreases the fastest for CFY, followed by CLY and then CFFY. The change in height of the peaks associated with cysteine-containing peptide (1001 cm−1, 1032 cm−1) and PVP (1770 cm−1) were fit with an asymptotic function. Raman spectra of the peptide powder were also obtained for peptides CFY and SFY (Supporting Information, S8). Spectra were also obtained for assorted peptide coated cubes and reference cubes on glass slides. A strong band in the range of 2550 to 2590 cm−1 is attributed to S−H36 and is observed in the CFY powder sample at 2585 cm−1. This band is not observed in the spectra of the cubes incubated in cysteinecontaining peptides (Supporting Information, S8), which supports the attachment of these peptides to the surface by a covalent silver−sulfur bond, since the S−H band is lost.
similar for the three peptides tested. This is encouraging as suggests that despite the fact that the films are likely multilayered, their properties could be fairly well controlled. Aggregation was not observed after incubation in SFY, and the peptide was not confirmed to be replacing PVP on the surface with SERS but rather attaching non covalently, indicating the importance of the thiol for attachment to the surface of the nanoparticles. It is likely that by varying the length and the primary structure (physicochemical properties) of the peptide applications of nanoparticles could be easily controlled.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b11708. Peptide Structures, calibration curves for peptides, additional TEMs for cysteine-containing peptides, AFM images of supernatant after incubation in 3 mM SFY, additional TEMs for cubes incubated in CFFY, energy dispersive X-ray spectra particles incubated in cysteinecontaining peptides and reference cubes, SERS of supported silver nanocubes incubated in 0.1 mM CFY, powder Raman spectra of CFY and SFY and SERS on substrates (PDF)
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AUTHOR INFORMATION
Corresponding Author
* Phone: (613) 520-2600 ext 6043; Fax: (613) 520-3749; Email:
[email protected]. ORCID
Anatoli Ianoul: 0000-0002-4933-9836 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support was provided by NSERC and OGS. ABBREVIATIONS CLY, cysteine-leucine-tyrosine; CFY, cysteine-phenylalaninetyrosine; CFFY, cysteine-phenylalanine-phenylalanine-tyrosine; SFY, serine-phenylalanine-tyrosine; PVP, poly(vinylpyrrolidone); SERS, surface enhanced Raman scattering; LSPR, localized surface plasmon resonance; TEM, transmission electron micrograph; EDX, energy dispersive X-ray spectra
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REFERENCES
(1) Xia, Y.; Xiong, Y.; Lim, B.; Skrabalak, S. E. Shape-Controlled Synthesis of Metal Nanocrystals: Simple Chemistry Meets Complex Physics? Angew. Chem., Int. Ed. 2009, 48, 60−103. (2) Wiley, B.; Sun, Y.; Mayers, B.; Xia, Y. Shape-Controlled Synthesis of Metal Nanostructures: The Case of Silver. Chem. - Eur. J. 2005, 11, 454−463. (3) De Palma, R.; Peeters, S.; Van Bael, M. J.; Van denRul, H.; Bonroy, K.; Laureyn, W.; Mullens, J.; Borghs, G.; Maes, G. Silane Ligand Exchange to Make Hydrophobic Superparamagnetic. Chem. Mater. 2007, 19, 1821−1831. (4) Hong, R.; Fischer, N. O.; Emrick, T.; Rotello, V. M. Surface PEGylation and Ligand Exchange Chemistry of FePt Nanoparticles for Biological Applications. Chem. Mater. 2005, 17, 4617−4621. (5) Parween, S.; Ali, A.; Chauhan, V. S. Non-Natural Amino Acids Containing Peptide-Capped Gold Nanoparticles for Drug Delivery Application. ACS Appl. Mater. Interfaces 2013, 5, 6484−6493.
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CONCLUSIONS In conclusion, the cysteine-containing peptides, CFY, CFFY and CLY, were able to replace PVP on the surface of silver nanocubes in solution as well as when supported on glass slides, which was confirmed with SERS, TEM, EDX and UV−vis. For the range of initial concentrations used in this work (1.5−8.5 mM), there was no observed increase in the resulting peptide layer density or thickness with increasing concentration for the cysteine containing peptides suggesting saturation of surface binding sites. At the same time, it is likely that the peptides form a multilayer structure around the nanocubes leading to aggregation of the particles in solution. The film properties are G
DOI: 10.1021/acs.jpcb.6b11708 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B (6) Briggs, B. D.; Li, Y.; Swihart, M. T.; Knecht, M. R. Reductant and Sequence Effects on the Morphology and Catalytic Activity of PeptideCapped Au Nanoparticles. ACS Appl. Mater. Interfaces 2015, 7, 8843− 8851. (7) Vignoni, M.; de Alwis Weerasekera, H.; Simpson, M. J.; Phopase, J.; Mah, T.-F.; Griffith, M.; Alarcon, E. I.; Scaiano, J. C. LL37 Peptide@silver Nanoparticles: Combining the Best of the Two Worlds for Skin Infection Control. Nanoscale 2014, 6, 5725−5728. (8) Chen, W.-Y.; Chang, H.-Y.; Lu, J.-K.; Huang, Y.-C.; Harroun, S. G.; Tseng, Y.-T.; Li, Y.-J.; Huang, C.-C.; Chang, H.-T. Self-Assembly of Antimicrobial Peptides on Gold Nanodots: Against MultidrugResistant Bacteria and Wound-Healing Application. Adv. Funct. Mater. 2015, 25, 7189−7199. (9) Xiong, N.; Dong, X.-Y.; Zheng, J.; Liu, F.-F.; Sun, Y. Design of LVFFARK and LVFFARK-Functionalized Nanoparticles for Inhibiting Amyloid B-Protein Fibrillation and Cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 5650−5662. (10) Liu, Y.; Wang, S.; Zhang, C.; Su, X.; Huang, S.; Zhao, M. Enhancing the Selectivity of Enzyme Detection by Using Tailor-Made Nanoparticles. Anal. Chem. 2013, 85, 4853−4857. (11) Chen, P.; Selegård, R.; Aili, D.; Liedberg, B. Peptide Functionalized Gold Nanoparticles for Colorimetric Detection of Matrilysin (MMP-7) Activity. Nanoscale 2013, 5, 8973−8976. (12) Lévy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. Rational and Combinatorial Design of Peptide Capping Ligands for Gold Nanoparticles. J. Am. Chem. Soc. 2004, 126, 10076−10084. (13) Palafox-Hernandez, J. P.; Tang, Z.; Hughes, Z. E.; Li, Y.; Swihart, M. T.; Prasad, P. N.; Walsh, T. R.; Knecht, M. R. Comparative Study of Materials-Binding Peptide Interactions with Gold and Silver Surfaces and Nanostructures: A Thermodynamic Basis for Biological Selectivity of Inorganic Materials. Chem. Mater. 2014, 26, 4960−4969. (14) Joshi, S.; Ghosh, I.; Pokhrel, S.; Mädler, L.; Nau, W. Interactions of Amino Acids and Polypeptides with Metal Oxide Nanoparticles Probed by Fluorescent Indicator Adsorption and Displacement. ACS Nano 2012, 6, 5668−5679. (15) Puddu, V.; Perry, C. C. Peptide Adsorption on Silica Nanoparticles: Evidence of Hydrophobic Interactions. ACS Nano 2012, 6, 6356−6363. (16) Doty, R. C.; Tshikhudo, T. R.; Brust, M.; Fernig, D. G.; Li, V.; Li, V. L.; Kingdom, U.; April, R. V.; Re, V.; Recei, M.; et al. Extremely Stable Water-Soluble Ag Nanoparticles. Chem. Mater. 2005, 17, 4630− 4635. (17) Fu, X.; Wang, Y.; Huang, L.; Sha, Y.; Gui, L.; Lai, L.; Tang, Y. Assemblies of Metal Nanoparticles and Self-Assembled Peptide FibrilsFormation of Double Helical and Single-Chain Arrays of Metal Nanoparticles. Adv. Mater. 2003, 15, 902−906. (18) Panda, J. J.; Chauhan, V. S. Short Peptide Based Self-Assembled Nanostructures: Implications in Drug Delivery and Tissue Engineering. Polym. Chem. 2014, 5, 4418−4436. (19) Tena-Solsona, M.; Escuder, B.; Miravet, J. F.; Casttelleto, V.; Hamley, I. W.; Dehsorkhi, A. Thermodynamic and Kinetic Study of the Fibrillization of a Family of Tetrapeptides and Its Application to Self-Sorting. What Takes So Long? Chem. Mater. 2015, 27, 3358− 3365. (20) Nanda, J.; Adhikari, B. Formation of Hybrid Hydrogels Consisting of Tripeptide and Different Silver Nanoparticle-Capped Ligands: Modulation of the Mechanical Strength of Gel Phase Materials. J. Phys. Chem. B 2012, 116, 12235−12244. (21) Sherry, L. J.; Chang, S.-H.; Schatz, G. C.; Van Duyne, R. P.; Wiley, B. J.; Xia, Y. Localized Surface Plasmon Resonance Spectroscopy of Single Silver Nanocubes. Nano Lett. 2005, 5, 2034−2038. (22) Bottomley, A.; Prezgot, D.; Ianoul, A. Plasmonic Properties of Silver Nanocube Monolayers on High Refractive Index Substrates. Appl. Phys. A: Mater. Sci. Process. 2012, 109, 869−872. (23) Zhang, S.; Bao, K.; Halas, N. J.; Xu, H.; Nordlander, P. Substrate-Induced Fano Resonances of a Plasmonic Nanocube: A Route to Increased-Sensitivity Localized Surface Plasmon Resonance Sensors Revealed. Nano Lett. 2011, 11, 1657−1663.
(24) Bottomley, A.; Prezgot, D.; Coyle, J. P.; Ianoul, A. Dynamics of Nanocubes Embedding into Polymer Films Investigated via Spatially Resolved Plasmon Modes. Nanoscale 2016, 8, 11168−11176. (25) Moran, C. H.; Rycenga, M.; Zhang, Q.; Xia, Y. Replacement of Poly(vinyl Pyrrolidone) by Thiols: A Systematic Study of Ag Nanocube Functionalization by Surface-Enhanced Raman Scattering. J. Phys. Chem. C 2011, 115, 21852−21857. (26) Borodko, Y.; Habas, S. E.; Koebel, M.; Yang, P.; Frei, H.; Somorjai, G. A. Probing the Interaction of Poly (vinylpyrrolidone) with Platinum Nanocrystals by UV - Raman and FTIR. J. Phys. Chem. B 2006, 110, 23052−23059. (27) Borodko, Y.; Humphrey, S. M.; Tilley, T. D.; Frei, H.; Somorjai, G. a. Charge-Transfer Interaction of Poly(vinylpyrrolidone) with Platinum and Rhodium Nanoparticles. J. Phys. Chem. C 2007, 111, 6288−6295. (28) Zhu, G.; Zhu, X.; Fan, Q.; Wan, X. Raman Spectra of Amino Acids and Their Aqueous Solutions. Spectrochim. Acta, Part A 2011, 78, 1187−1195. (29) Eisenberg, D.; Weiss, R. M.; Terwilliger, T. C.; Wilcox, W. Hydrophobic Moments and Protein Structure. Faraday Symp. Chem. Soc. 1982, 17, 109−120. (30) Asher, S. A.; Ludwig, M.; Johnson, C. R. UV Resonance Raman Excitation Profiles of the Aromatic Amino Acids. J. Am. Chem. Soc. 1986, 108, 3186−3197. (31) Dudik, J. M.; Johnson, C. R.; Asher, S. A. UV Resonance Raman Studies Models for Peptide Bond. J. Phys. Chem. 1985, 89, 3805−3814. (32) Wiley, B. J.; Hyuk, I. S.; Li, Z.-Y.; McLellan, J.; Siekkinen, A.; Xia, Y. Maneuvering the Surface Plasmon Resonance of Silver Nanostructures through Shape-Controlled Synthesis. J. Phys. Chem. Chem. B 2006, 110, 15666−15675. (33) Mahmoud, M. A.; Tabor, C. E.; El-Sayed, M. A. SurfaceEnhanced Raman Scattering Enhancement by Aggregated Silver Nanocube Monolayers Assembled by the Langmuir - Blodgett Technique at Different Surface Pressures. J. Phys. Chem. C 2009, 113, 5493−5501. (34) Fischer, S.; Papageorgiou, A. L-Cysteine on Ag (111): A Combined STM and X-Ray Spectroscopy Study of Anchorage and Deprotonation. J.Phys. Chem.C 2012, 116, 20356−20362. (35) Bolduc, O. R.; Clouthier, C. M.; Pelletier, J. N.; Masson, J.-F. Peptide Self-Assembled Monolayers for Label-Free and Unamplified Surface Plasmon Resonance Biosensing in Crude Cell Lysate. Anal. Chem. 2009, 81, 6779−6788. (36) Diaz Fleming, G.; Finnerty, J. J.; Campos-Vallette, M.; Célis, F.; Aliaga, A. E.; Fredes, C.; Koch, R. Experimental and Theoretical Raman and Surface-Enhanced Raman Scattering Study of Cysteine. J. Raman Spectrosc. 2009, 40, 632−638.
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DOI: 10.1021/acs.jpcb.6b11708 J. Phys. Chem. B XXXX, XXX, XXX−XXX
