Tsukasa
Mizutaru
a,
Taro
Sakuraba
a,
Toru
Nakayama
a,
Galina
Marzun
b,
Philipp
Wagener
b,
Christoph
Rehbock
b,
Stephan
Barcikowski
b,
Katsuhisa
Murakami
cd,
Junichi
Fujita
cd,
Noriyuki
Ishii
e and
Yohei
Yamamoto
*ad
aDivision of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan. E-mail: yamamoto@ims.tsukuba.ac.jp
bTechnical Chemistry I and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, NanoEnergieTechnikZentrum (NETZ), Carl-Benz-Strasse 199, 47057 Duisburg, Germany
cDivision of Applied Physics, Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan
dTsukuba Research Center for Interdisciplinary Materials Science (TIMS), Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan
eNational Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
First published on 16th July 2015
Oligopeptide β-sheets comprising a fluorenyl methoxy carbonyl (Fmoc) group on its N-terminus and five amino acid residues of cysteine, lysine and valine displays redispersive properties with respect to agglomerated metal nanoparticles (MNPs, M = Au, Cu, Pt and Pd). The ligand-free MNPs prepared by a laser ablation technique in liquid maintain a high dispersion state due to the inherent surface charges delivered by anionic species present in solution, but may agglomerate after the preparation depending on concentration or salinity. We show how the agglomerated MNPs can be returned to the dispersed state by adding the Fmoc-oligopeptide β-sheets in methanol, as characterized by photoabsorption spectroscopy and transmission electron microscopy. Systematic studies in which we vary the concentration, the amino acid sequences and the secondary structures of a series of the oligopeptides clarify that the β-sheet structure is essential for the redispersion of the MNPs, where metal-binding thiol groups are integrated on one side and positively charged amino groups are located on the other side of the β-sheet. A possible mechanism for the redispersion may be that the agglomerated MNPs are subsequently enwrapped by the flexible β-sheets and gradually separated due to the reconstruction of peptide β-sheets under the assembly/disassembly equilibrium.
Especially, when the nanoparticles are small, the high surface energy of the particles can cause agglomeration. As the size of the particles decreases, the interaction forces between particles increases, resulting in a loss of their specific properties. Furthermore, once the agglomeration occurs, it is rather difficult to redisperse nanoparticles to their original dispersed state.8
Ligand free MNPs in aqueous or organic solvents can be produced by pulsed laser ablation in liquids.9 The irradiation with a focused high energy density laser beam of a metal target in a solvent results in the production of highly dispersed MNPs with diameters of several to several tens of nanometres. Because the resulting MNPs are charged and their surfaces are covered with anionic species that are present in solution, a repulsive electrostatic force exists between the particles. Accordingly, the MNPs maintain their highly dispersed colloidal state in solutions of micromolar salinity and at moderate particle concentrations.9 However, in pure solvents or at high concentrations, the stability of the dispersed MNPs gradually diminishes and several months after the preparation of the colloidal solution, agglomeration of the MNPs might occur. In such cases, the plasmonic characters of the MNPs are altered due to plasmon coupling, inducing a red shift of the surface plasmon resonance (SPR) band and an increased scattering in the near infrared (NIR) spectral regime.10 Therefore, if the agglomerated MNPs can be redispersed efficiently, the plasmonic property will be recovered to the original state, and the MNPs can be utilized as renewable materials, which are beneficial from an ecological and sustainable point of view.
Redispersion may be done via ultrasonification in a solvent, where the liquid is oscillating due to the energy transfer and causes nucleation and collapse of solvent bubbles. Then agglomerates can be broken due to the bubble collapse on the solid surfaces. Deagglomeration is then influenced by the input energy and time. However, reagglomeration can take place in case the particles are not sufficiently stabilized. Furthermore abrasion of the ultrasonic horn can occur, which may cause unwanted contaminations.
In this study, we developed redispersants for agglomerated MNPs, and found that a short oligopeptide with a certain amino acid sequence can redisperse them very efficiently. The secondary structure of the oligopeptides (β-sheet structure) plays an important role for the redispersion of MNPs. When peptides form a β-sheet, the side chains of the amino acids protrude alternately on the upper and lower sides of the β-sheet.11 The redispersive oligopeptides include one cysteine (C) and one lysine (K) residue. Cysteine has a thiol side chain that tends to bind with metals, while lysine has an amino group on the side chain which is positively charged in a neutral aqueous solution. As a result of the β-sheet formation, metal-binding with thiol side chains predominantly occurs on one side of the β-sheet, while the positive charges are located on the other side. These bilateral properties of the peptide β-sheet may be responsible for redispersing agglomerated MNPs. A fluorenyl methoxy carbonyl (Fmoc) group attached on the N-terminus of the oligopeptides plays a role in facilitating the β-sheet formation of the peptide.
The products were subjected to recycling preparative HPLC (Japan Analytical Industry model LC-9210 II NEXT recycling preparative HPLC equipped with a JAIGEL-ODS-AP-A column) with MeOH/TFA (100/0.1) as an eluent, where the major fraction was collected and evaporated to allow isolation of the corresponding R-oligopeptide as white solid. The final products were characterized by MALDI-TOF MS spectrometry on an AB SCIEX model TOF/TOF(TM) 5800 system spectrometer using CHCA as a matrix. The synthesized Fmoc-peptide, for example, Fmoc-VKVVC as representative, is soluble in MeOH, EtOH and dimethyl sulfoxide, while solubility in water, acetone and chloroform is low.
Photoluminescence (PL) spectra were measured at 25 °C with a JASCO model FP-6500 spectrofluorometer. For sample preparation, MeOH solutions of tris(8-hydroxyquinoline)aluminum (Alq3, 0.22 mM, 1 mL) and its mixture with self-assembled Fmoc-VKVVC in MeOH (0.5 mM, total amount of 1 mL) were added to aqueous solutions of AuNPs (1 mL). A 10 mm path length quartz cuvette was used for PL measurements with the excitation wavelength, excitation and emission band widths, response and sensitivity factors set at 390 nm, 3 nm, 3 nm, 0.02 s and medium, respectively. The scan rate and the number of integration were 2000 nm min−1 and 10, respectively. In addition, a MeOH/water mixed solution of Alq3 was prepared and measured as a control.
For propensity calculation of the β-sheet formation, the TANGO algorithm program was utilized, which was freely downloaded from the internet at http://tango.embl.de/.12 The calculation was carried out under the following conditions; pH = 8.5, T = 298.15 K, and ionic strength = 0.004.
For redispersion experiments, MeOH solutions of oligopeptides (0.5 mM), which had been incubated for 7 days, were added to the MNP dispersion (1/1 v/v). At this peptide concentration, the amount of added molecules per nanoparticle is about 407, which corresponds to ∼1 peptide per surface atom assuming an average particle size of 5 nm. The mixtures were kept for 3 days in dark in order to avoid the effect of light. The redispersion experiments using a MeOH solution of 1-dodecanethiol (C12H25SH) (0.5 mM) were carried out in the same way. Electronic photoabsorption spectra were recorded on a JASCO model UV-650 UV-vis-NIR spectrophotometer within a wavelength range of 300–900 nm.
The TEM micrograph of Fmoc-VKVVC, self-assembled in MeOH, displayed nanofibrillar structures with 10–30 nm width and sub-micrometer length (Fig. 1e). Such fibrillar morphology is typical of a peptide β-sheet. Considering the amino acid sequence and previous reports on the assembly of Fmoc-substituted oligopeptides,15 the β-sheet most likely adopts an antiparallel configuration, where the side chains of K (animo group) and C (thiol group) are located separately on the upper and lower sides of the β-sheet, respectively.
Fig. 2 (a) Photographs of the aqueous dispersion of AuNPs just after preparation (left), 6 months after preparation (centre) and finally 6 months after preparation, on the addition of a MeOH solution of self-assembled Fmoc-VKVVC and subsequent aging for 3 days (right). (b) TEM micrograph of agglomerated AuNPs in water. (c and d) TEM micrographs of agglomerated AuNPs after addition of MeOH solution containing Fmoc-VKVVC. (e) Electronic photoabsorption spectra of as-prepared AuNPs in water (dotted), agglomerated AuNPs in water (blue), and after addition of MeOH solution of Fmoc-C (green) and Fmoc-VKVVC (red) into the agglomerated AuNPs in water. The spectra were normalized by the interband absorption at 380 nm, where the absorbance is not influenced by the agglomerated state but is proportional to gold mass.9d (f) Photoemission spectra of MeOH/water (1/1 v/v) mixed solution of Alq3 (black), mixture of Alq3 and agglomerated AuNPs (blue), and Alq3 and AuNPs redispersed by self-assembled Fmoc-VKVVC (red). (g) Electronic photoabsorption spectra of agglomerated AuNPs (black) and that mixed with Fmoc-VKVVC in MeOH. Total concentrations of Fmoc-VKVVC in MeOH/water (1/1 v/v): 0.0025 (orange), 0.0625 (green), 0.125 (blue) and 0.25 mM (red). Inset shows plot of absorbance at 530 nm versus total concentration of Fmoc-VKVVC. (h) CD spectra of Fmoc-VKVVC in MeOH/water (1/1 v/v). Total concentrations of Fmoc-VKVVC: 0.0025 (orange), 0.0625 (green), 0.125 (blue) and 0.25 mM (red). Inset shows plot of CD intensity at 210 nm versus total concentration of Fmoc-VKVVC. |
When the MeOH solution of the β-sheets of Fmoc-VKVVC (0.5 mM) was added to the aqueous dispersion of the agglomerated AuNPs and the mixture was aged for 3 days, the colour of the dispersion gradually turned back to pink (Fig. 2a, right), which is analogous to the initial state of the AuNP dispersion in water. Electronic photoabsorption spectroscopy showed an apparent spectral change before and after addition of the MeOH solution of Fmoc-VKVVC. Before addition, the absorption maximum appeared at 630 nm with an absorption shoulder at 530 nm (Fig. 2e, blue). After addition, the peak at 630 nm completely disappeared, and the absorption at 530 nm due to the surface plasmon resonance of isolated AuNPs was observed (Fig. 2e, red). TEM micrographs after addition of Fmoc-VKVVC showed that each AuNP is clearly separated from its neighbouring particle with a certain distance of 1–5 nm (Fig. 2c and d). The presence of the underlying peptides may not be detectable due to the relatively weak electron beam scattering of organic matter in comparison to heavy elements like Au. However, in the wide range view, we can identify that dispersed AuNPs are attached separately to β-sheets (Fig. 2c, inset). The pink colour of the dispersion was maintained at least 9 months after addition of the MeOH solution of Fmoc-VKVVC.
AuNPs can act as a quencher for the fluorescence of dyes.16 This phenomenon is observed in systems where the dye serves as the donor and the plasmonic NP as the acceptor species. It is predominantly attributed to two separate effects. Firstly, non-radiative energy transfer similar to FRET is found, however a second radiative effect, originating from an out of phase oscillation of fluorophores and metal NP, was also observed.17 Consequently, efficient fluorescence quenching by metal NP may also occur where the spectral overlap between fluorescence emission and NP absorbance is relatively small, e.g., in case of the combination of AuNP and red emitting dyes like Cy5.17b In both cases, the fluorescence quenching effect is enhanced significantly when the distance between fluorophore and NP surface is decreased as well as when the particle size is increased.17b
We investigated the fluorescence quenching properties of the redispersed AuNPs. A MeOH/water mixed solution of Alq3 shows a PL band centered at 510 nm (Fig. 2f, black), which overlaps well with the plasmon absorption band of AuNPs (∼530 nm). Accordingly, fluorescence quenching by non-radiative effects is prone to occur in the observed system. In fact, 69% of the fluorescence from Alq3 was quenched when the AuNPs, redispersed by the β-sheets of Fmoc-VKVVC, were added to a solution containing Alq3 (Fig. 2f, red). The degree of quenching is much higher than in case of the original partially agglomerated AuNPs, where a quenching of ∼46% was observed (Fig. 2f, blue). In comparison to literature studies, where quenching rates >90% were observed, the values found in our study are comparably low. This is predominantly attributed to the fact that the Alq3 was not fixed on the NP surface and that the unbound dye molecules were not removed in this experimental setup. Nonetheless, a significantly more pronounced quenching was found for the dispersed particles in comparison to the aggregated ones. This may be due to the more intensive overlap between the SPR maximum of the particles and the dye's emission spectrum, which could enhance the non-radiative quenching rate. These experimental results could indicate that the plasmonic state of the redispersed AuNPs turned back to that of the initial state of the dispersed AuNPs. However, it should be noted that next to the plasmonic state, the total surface area of the AuNPs was also significantly increased in comparison to the agglomerated particles. Consequently, the higher abundance of surface area could lead to a higher number of binding events of the dye molecules to the metal surface, which could also result in a more pronounced quenching.
When the concentration of the added peptide solution was increased by two or three times, the incubation time required for the redispersion of agglomerated AuNPs was shortened to 1–2 days. On the other hand, when the concentration of the added peptide was reduced, a clear threshold for the redispersion of agglomerated AuNPs was observed. Fig. 2g shows the absorbance change upon addition of Fmoc-VKVVC in MeOH to the aqueous solution of agglomerated AuNPs. Less than 0.0625 mM of the total concentration of Fmoc-VKVVC resulted in much heavier agglomeration, leading to the decrease of the absorbance of AuNPs. This is probably attributed to the well-known charge compensation effect, previously described by Gamrad et al. for negatively-charged nanoparticles and positively-charged peptides.9g At a certain concentration regime, an isoelectric point is reached where charge compensation induces impairment of the colloidal stability. This tendency is similar to the aggregation of AuNPs when cysteine was added to aqueous dispersion of AuNPs.18 In contrast, when the total concentrations of Fmoc-VKVVC were increased to 0.125 and 0.25 mM, the absorbance of AuNPs at 530 nm was increased (Fig. 2g, inset). The CD spectra of Fmoc-VKVVC also show concentration dependency, where concentrations of Fmoc-VKVVC higher than 0.125 mM display negative ellipticity at 210 nm, while those less than 0.0625 mM show negligible ellipticity in that wavelength region (Fig. 2h). These spectroscopic results clearly indicate that the β-sheet formation of Fmoc-VKVVC is essential for the redispersion of agglomerated AuNPs.
As a control, addition of Fmoc-C in MeOH to the agglomerated AuNP aqueous dispersion hardly showed a spectral change (Fig. 2e, green).18 Furthermore, the addition of MeOH to freshly-prepared AuNPs in water did not result in significant changes in the absorption spectra. Therefore, we conclude that the redispersion phenomena are solely related to Fmoc-VKVVC.
We further investigated the redispersion properties of Fmoc-VKVVC with respect to other agglomerated MNPs (M = Cu, Pt and Pd). Before addition of the MeOH solution of Fmoc-VKVVC, the MNPs were heavily agglomerated (Fig. 3i). Upon addition of MeOH only or a MeOH solution of Fmoc-C, the agglomeration of MNPs intensified, and precipitation could be observed by eye (Fig. 3ii and iii). In contrast, upon addition of MeOH solutions of Fmoc-VKVVC, the precipitates were readily dispersed by a gentle stirring, and the mixed solutions became homogeneously coloured with high transparency (Fig. 3iv).
We further checked the redispersion properties of Fmoc-pentapeptides with different amino acid sequences, Fmoc-VVVKC and Fmoc-VVVVC. Fmoc-VVVKC did not form β-sheet in MeOH, and hardly redispersed the agglomerated AuNPs (Fig. 4f). Fmoc-VVVVC itself was hardly dissolved in MeOH and formed a macroscopic precipitation, and the dispersion of AuNPs was significantly worsened by adding the MeOH suspension containing Fmoc-VVVVC (Fig. 4g).
For comparison, MeOH solutions of Fmoc-VKVVE9c and Fmoc-VKVVH, which contain E (negatively charged residue) or H (metal-ion binding residue) instead of C (metal-binding residue), were added to the agglomerated AuNP dispersion. Fmoc-VKVVE formed a β-sheet structure in MeOH,9c while Fmoc-VKVVH hardly formed β-sheet. Both of these Fmoc-peptides in MeOH did not redisperse agglomerated AuNPs (Fig. 4h and i). Accordingly, metal-binding C seems to be one of the critical structural components for redispersing agglomerated MNPs. We also investigated the redispersion properties in the presence of the non-peptide compound 1-dodecanethiol, which includes a metal-binding thiol group. However, the AuNPs in water agglomerated much more severely upon the addition of a MeOH solution of 1-dodecanethiol, though these effects were probably increased by the poor solubility of 1-dodecanethiol in aqueous environments. (Fig. 4j).
Sequence & structure | Assembly in MeOH | Dispersion propertya |
---|---|---|
a ○; highly dispersive, ×; hardly dispersive. b Ref. 11c. | ||
Fmoc-VKVVC | β-Sheet | ○ |
H-VKVVC | Random | × |
Fmoc-KVVC | Random | × |
Fmoc-VVC | Random | × |
Fmoc-VC | Random | × |
Fmoc-C | Random | × |
Fmoc-KC | Random | × |
Fmoc-VVVKC | Random | × |
Fmoc-VVVVC | Insoluble | × |
Fmoc-VKVVE | β-Sheetb | × |
Fmoc-VKVVH | Random | × |
1-Dodecanethiol | Soluble | × |
Fig. 5 shows a schematic representation of the possible mechanism for β-sheet redispersion of the agglomerated MNPs. β-Sheets of Fmoc-VKVVC possess highly integrated thiol groups on one side. When a MeOH solution of the β-sheet is added to the dispersion of the agglomerated MNPs, the β-sheet subsequently enwraps the MNPs (Fig. 5b). Dissociation of the agglomerated MNPs possibly takes place in the equilibrium of association (β-sheet formation)/dissociation process of Fmoc-VKVVC in solution. Once the MNPs are dissociated, they will be bound on one side of the β-sheet (Fig. 5c). The β-sheet of Fmoc-VKVVC is dispersive in aqueous solution because of the positive charges of K on the other side of the β-sheet, resulting in the highly dispersed MNPs as a hybrid material. In contrast, Fmoc-VVVKC hardly redispersed agglomerated MNPs, possibly because of the lack of the β-sheet secondary structure.
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