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The precision spectral features of tryptophan-tryptophan (Trp-Trp) dipeptides on PSSS-templated silver nanoparticles (Ag NPs) were investigated by combining UV-visible absorption, surface enhanced Raman scattering (SERS), and fluorescence spectroscopy. The secondary surface plasmonic absorption band suggested the existence of the Ag NP dimer, bridged by two indole rings in Trp-Trp dipeptides. The proposed π-π stacking interaction provided excellent SERS spectrum showing the adsorption sites are indole ring moieties. The fluorescence intensity of Trp-Trp dipeptides was quenched by almost 30% with 0.5 ns increase in lifetime, due to charge transfer in the Trp-Trp dipeptides/silver system. The 0.5 ns lifetime was independent of emission wavelength.
© 2015 Optical Society of America
1. Introduction
Noble silver nanoparticles (Ag NPs) have received increased interest in recent years, due to their unique chemical, physical properties, which are distinct from their bulk scale [1]. One of their important physical properties is that the electronic field intensity near the silver surface is significantly enhanced relative to the incident light field. The enhancement mainly originates from surface plasmonic resonance (SPR), i.e., the collective excitation of conductor electrons in the metal surface produces a resonance at certain frequencies of excitation photons [2–4 ]. Two important field enhancement applications are surface enhanced fluorescence (SEF) [5–7 ] and surface enhanced Raman scattering (SERS) spectroscopy [8–12 ].
Amino acids are the building blocks of proteins and enzymes, and various amino acid residues produce numerous peptides by dehydration condensation reactions. Peptide interactions with nano-metal surfaces play an important role in the efficiency of bio-analytical applications; compatibility of biosensors; and explanation of adsorption, adhesion, and orientation of peptides [13–17 ]. In light of the importance of the peptide/metal surface system, it is of considerable significance to study the complex system
Trp-Trp dipeptides comprise two tryptophan residues linked by a peptide bond, which indicates that Trp-Trp dipeptides molecules have better flexibility than single tryptophan molecules [18]. The Trp-Trp dipeptides/silver system is a prototype model to study interactions between peptides and the silver surface with the indole ring providing high sensitivity to local environmental variation [19], high quantum yield [20], sizable Raman scattering cross sections, [REMOVED HYPERLINK FIELD]and a wealth of analogues [21].
Numerous researchers [22–25 ] have demonstrated that SERS spectroscopy is a useful analytical tool to characterize interactions of tryptophan and its dipeptides on silver surfaces. There are three possible adsorption sites: (a) via carboxylate moiety; (b) via amino moiety; (c) via indole ring (active N atom, π electron system). However, there remains some controversy regarding these absorption sites and related peptides. Herne et al. [15] found that Trp-Trp dipeptides molecules interact with silver surface only through the amine group. However, Stewart et al. [21] argued that, owing to the protonation of the amine terminal, the absorption site was via the ionized carboxylate group. Lee [26] and Aliaga [27] showed that interaction sites of tryptophan molecules on silver surfaces were via carboxylate and amine moieties, and Lee et al. [26] also suggested the possibility that the indole ring moiety binds to the silver surface.
Although SERS spectroscopy can efficiently determinate the orientations of tryptophan and related peptides on the silver surface, influencing fluorescence emissions from interactions of adsorbates with the silver surface remains unknown. Fortunately, fluorescence emission intensity and lifetime can provide valuable information regarding the interaction mechanisms of the Trp-Trp dipeptides/silver system.
We designed Poly (sodium −4- styrenesulfonate) (PSSS)-templated Ag NPs substrates, which were used to study the spectral features of Trp-Trp dipeptides. The PSSS-templated Ag NPs have high affinity to Trp-Trp dipeptides molecules via the π-π stacking interaction. High quality SERS spectra of Trp-Trp dipeptides show that the interaction sites on the silver surface are indole rings. From fluorescence measurements, the charge transfer between the indole ring chromophore and the silver surface quenched the fluorescence intensity of Trp-Trp dipeptides, while long lifetime increased by almost 0.5 ns. The critical spectral features are expected to provide a valuable database for further studies of similar peptides and proteins in life science.
2. Experiments
2.1 Materials
Tryptophan-tryptophan dipeptides (99%), L-tryptophan (≥99.5%, Bio-ultra), PSSS (MW: 1000000) were purchased from Sigma-Aldrich. Silver nitrate, sodium borohydride, and trisodium citrate were purchased from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water with resistance of ~18.2 MΩ was used for all the experiments. All the reagents were used without any further purification.
2.2 Synthesis of Ag NPs
The PSSS-templated Ag NPs were prepared by reduction of silver nitrate in a solution of PSSS (250 μL 500 mg/L), freshly prepared sodium borohydride (300 μL 10 mM), and trisodium citrate (5 mL 2.5 mM). The silver nitrate solution (5 mL 0.5 mM) was injected into the mixture over two minutes while vigorous stirring. After approximately 3 minutes the synthesis was complete.
Spherical Ag NPs were synthesized following Creighton’s procedure [28] with some modifications. In an ice-bath, silver nitrate solution (10 ml 1 mM) was injected into freshly prepared sodium borohydride solution (30 ml 2 mM) drop by drop while stirring the mixture solutions vigorously. Once the synthesis was completed, the surface plasmonic absorption (SPA) band of Ag spheres was identified at approximately 392 nm.
2.3 UV-Visible absorption and transmission electron microscopy
The extinction spectra of Ag NPs in the spectral range 200-800 nm were measured by a two-beam UV-Visible spectrometer (TU-1901, China). Transmission electron microscopy (TEM) images of the PSSS-templated Ag NPs were obtained from a HT 7700 system (Hitachi, Japan) at 100 kV.
2.4 Spectra measurements
Raman spectra of Trp-Trp dipeptides were recorded with a confocal microscope Raman system (Jobin-Yvon T64000, spectral resolution of 2 cm−1) equipped with 532 nm exciting laser, highly sensitive liquid-N2 cooled charge-coupled detector, and 600 g/mm holographic grating. A notch filter was used to eliminate Rayleigh scattering. The Raman spectrometer system was calibrated with silicon wafer for the Raman band centered at 520 cm−1.
Steady-state fluorescence spectra were measured with a Hitachi F-4500 spectro-fluorometer. The excitation wavelength was 295 nm, and slit bandwidth of excitation and emission was 2 nm. Under magic-angle conditions, time resolved fluorescence was obtained by time correlated single photon counting. The excitation source was a pulsed diode laser (PicoQuant, Germany) with the central wavelength at 295 nm and repetition frequency at several MHz. A photomultiplier tube (PMT) was employed to detect the time resolved fluorescence spectrum. A OD 4 long-pass filter was employed to remove excitation light scattering, and a monochromator with slit width 1 mm selected the desired fluorescence wavelength. The instrument response function (IRF) was calculated from Rayleigh scattering of silica aqueous solution. Fluorescent decays of the samples were recorded to 10,000 counts at the peak channel. Decay associated emission spectra (DAS) were recorded at 345 to 375 nm with 5 nm increment. The lifetimes of Trp-Trp dipeptides were obtained by fitting the decay curves to a three exponent model, and fit quality was assessed using the chi-squared test.
3. Results and discussion
3.1 Characterization of PSSS-templated Ag NPs
The morphology of PSSS-templated Ag NPs was characterized by TEM imaging. The PSSS-templated Ag NPs have an intense surface plasmon (SPA) band at 398 nm as shown in curve a in Fig. 1(a) , with irregular shape in Fig. 1(b), indicating that there are unexpected adsorption sites and enhancement hotspots. When the Ag NPs were mixed with Trp-Trp dipeptides solution, significant variation of the SPA bands as shown in curve b in Fig. 1(a) shows strong interaction between PSSS-templated Ag NPs and Trp-Trp dipeptides. Although the primary SPA band was red-shift from 398 nm to 405 nm, the band was still intense as shown in curve b in Fig. 1(Aa. A new secondary SPA band developed at approximately 480 nm. This secondary band suggests that some Ag NPs have a new shape. Variations in solution pH and interactions of PSSS-templated Ag NPs with Trp-Trp dipeptides molecules can induce secondary SPA bands. To identify the cause, we also measured the extinction spectra of PSSS-templated Ag NPs under different conditions in Fig. 1(c). Since the pH value of the mixture solutions composed of Trp-Trp dipeptides and Ag NPs was approximately 6.0, the pH value of silver colloids was adjusted to 6.0 by addition of phosphate buffer. Comparing with the SPA band of freshly prepared Ag NPs, the SPA band in phosphate buffer produced no significant change, aside from a lower absorbance. Thus, the secondary SPA band is attributed to the strong interaction between PSSS-templated Ag NPs and Trp-Trp dipeptides molecules.
Fig. 1 (A) The extinction spectra of fresh freshly prepared PSSS-templated Ag NPs (a), after adding 1 mM Trp-Trp dipeptides 1 hour later (b). The inset in Fig. 1(a) is the color of silver colloids; (B) The TEM imaging of PSSS-templated Ag NPs; (C) The extinction spectra of PSSS-templated Ag NPs (a), after adding pH 6.0 phosphate buffer 1 hour later (b); (D) The extinction spectra of fresh freshly prepared PSSS-templated Ag NPs (a), after adding 1 mM Tryptophan solution 1 hour later (b).
Unlike Trp-Trp dipeptides molecules, the SPA band of PSSS-templated Ag NPs with tryptophan molecules adsorbed remains in Fig. 1(d). Therefore, the secondary band at about 480 nm is most likely due to SP absorption of the Ag NP dimer. The PSSS-templated Ag NPs wrapped aromatic moieties can easily adsorb indole rings of Trp-Trp dipeptides via π-π stacking interactions. It is very possible that two indole rings (π electron systems) of Trp-Trp dipeptide molecules spontaneously bridge a pair of neighbored Ag NPs, and then form the Ag NPs dimer. A great advantage of Ag NP dimer bridged by flexible molecules is that the dimer gap can also provide enhanced hotspots to improve the intensity of the SERS signals of adsorbed molecules [29,30 ].
Considering the possible influence of the pH value on the regulating SPA band from Ag NPs, we further measured the SPA bands of mixture solutions composed of PSSS-templated Ag NPs and Trp-Trp dipeptides solutions in different pH conditions, as shown in Fig. (2) . The SPA band (indole ring moiety, 280 nm) of Trp-Trp dipeptides was not significantly altered by pH value. Moreover, the SPA band at long wavelength bands (particularly at 480 nm resulting from the absorption of Ag NP dimers) is almost independent of the pH value (6.0-8.0). These results indirectly demonstrate that the adsorption moiety of Trp-Trp dipeptides on PSSS-templated Ag NPs is mainly via indole ring sites.
Fig. 2 Normalized extinction spectra of mixture solutions composed of PSSS-templated Ag NPs and Trp-Trp dipeptides solutions in various pH conditions. The inset is SPA bands of Ag NPs induced by Trp-Trp dipeptides.
3.2 SERS spectra of Trp and Trp-Trp dipeptides
Prior to characterizing the proposed π-π stacking interaction mechanism, the SERS spectrum for tryptophan molecules on PSSS-templated Ag NPs was measured, as shown in Fig. 3(a) . At detection level 500 μM, Raman signals of tryptophan are barely detected. However, for silver spheres with similar SPA band in Fig. 3(b), the SERS spectrum of tryptophan molecules (10−5 M) is easily obtained in Fig. 3 (c). The weaker SERS signals of tryptophan molecules on PSSS-templated Ag NPs show that the tryptophan molecules prefer to lie flat on the silver surface. In the parallel orientation mode, the enhanced electronic field near the silver surface does not align with transition dipole moments for the in-plane vibration modes. Therefore, the in-plane vibrations of indole ring cannot be efficiently enhanced.
Fig. 3 (A) SERS spectrum of L-tryptophan molecules (0.5 mM) on PSSS-templated Ag NPs. (B) Extinction spectra of fresh freshly prepared Ag spheres. Inset shows the color of Creighton silver colloids. (C) SERS spectrum of L-tryptophan molecules (10−2 mM) on Creighton silver colloids (D) (a) Normalized normal Raman spectrum of solid Trp-Trp dipeptides powder; (b) Normalized Raman spectrum (FWHM = 8 cm−1) of Trp-Trp dipeptides by DFT simulation; (c) Normalized SERS spectrum of (500 μM)Trp-Trp dipeptides on PSSS-templated Ag NPs.
However, when the Trp-Trp dipeptides molecules were chosen as target molecules, high quality SERS spectrum was acquired. The SERS and normal Raman (NR) spectra of Trp-Trp dipeptides are shown in Fig. 3(d). The Raman bands of indole ring vibrations dominate in the NR and SERS spectra. The most related bands of indole ring as shown in curve a in Fig. 3(d), are observed at 759, 879, 1013, 1342, 1361, and 1551 cm−1, respectively. The band at 1425 cm−1 is attributed to carboxylate group while the band at 1249 cm−1 corresponds to amino moiety. The enhanced vibration bands of the indole ring as shown in curve c in Fig. 3(d), are present at 740, 839, 1340, and 1573 cm−1. The Raman bands at 1249 cm−1 (for amino moiety) and 1425 cm−1 (for carboxylate moiety) were almost completely suppressed. The two strongest Raman bands were attributed to indole ring moiety. According to previous SERS reports on tryptophan and related dipepetides [15,21,26,27 ], when NH2 or COOH moiety interacts with Ag NPs, the Raman vibration bands assigned to NH2 or COOH moiety will be largely enhanced. For Trp-Trp dipepetides, there are three possible adsorption sites: (a) via carboxylate moiety; (b) via amino moiety; (c) via indole ring. The Raman vibrations from carboxylate and amino moieties are attentuated, while the indole ring vibrations are relatively amplified as shown in curve c in Fig. 3(d). It is proposed that there is π-π stacking interaction mechanism between Trp-Trp dipeptides and PSSS templated Ag NPs.
In contrast with strongly enhanced out-of-plane vibrations of the indole ring (1361, and 1551 cm−1), the Raman band at 1013 cm−1, ascribed to in-plane breathing vibrations of indole ring, disappears, and the other bands at 740, and 839 cm−1 are relatively less enhanced. The strong band at 759 and 879 cm−1 in the NR spectrum appear at 740, and 839 cm−1 in SERS spectrum, frequency shifts of 19 and 40 cm−1, respectively, which argues that there are strong interactions between PSSS-templated Ag NPs and the indole ring. This spectral specificity arises from the interaction of the indole ring with the silver surface when the tryptophan residue locates at the C-terminus position in a peptide, which is in good agreement with previously published observations [26].
In order to rule out the adsorption possibilities of carboxylate and amino moieties on PSSS templated Ag NPs, we measured the SERS spectrum of another typical tryptophan dipepetides (N-Acetyl-tryptophan-tryptophan methylester, NATrp2ME), as illustrated in Fig. 4 . Because of its C-terminus and N-terminus with weak polarity, it is unlikely that NATrp2ME molecules interact with Ag NPs via carboxylate and amino moieties. The main enhanced bands at 665, 1354, 1585 cm−1 belong to the Raman vibrations of indole ring moiety. It provides another experimental evidence to support our proposal.
Fig. 4 SERS spectrum of tryptophan dipepetides (N-Acetyl-tryptophan-tryptophan methylester, NATrp2ME, 50 μM) on PSSS-templated Ag NPs. The inset is chemical structure of NATrp2ME.
Therefore, the π-π stacking interaction mechanism of Trp-Trp dipeptides on PSSS-templated Ag NPs is verified.
3.3 Fluorescence spectra of Trp-Trp dipeptides in PSSS-templated Ag NPs
Although the π-π stacking interaction in Trp-Trp dipeptides/ PSSS-templated Ag NPs system was confirmed by SERS spectroscopy, there is no detailed information on fluorescence emission of Trp-Trp dipeptides. Fluorescence spectroscopy supplements our study of the Trp-Trp dipeptides in the complex system. The stead state fluorescence of Trp-Trp dipeptides adsorbed on PSSS-templated Ag NPs was measured, as shown in Fig. 5(a) . Compared with the Trp-Trp dipeptides in phosphate buffer, the fluorescence intensity in silver colloids was quenched by almost 30%, with a blue-shift of 5 nm. Due to the short distance from adsorbed Trp-Trp dipeptides to the silver surface, it is reasonable that the interaction has the ability to quench the fluorescence intensity of Trp-Trp dipeptides. In addition, via π-π stacking interaction mechanism, the fluorescence intensity of tryptophan adsorbed on PSSS-templated Ag NPs was quenched significantly, as well as 10 nm blue-shift in emission location, as shown in Fig. 5(b). The similar phenomenon provide another evidence to evaluate our proposal that tryptophan lies flat on silver surface of PSSS-templated Ag NPs.
Fig. 5 (A) Steady state fluorescence emission spectra (a) 0.5 mM Trp-Trp dipeptides in phosphate buffer (pH 6.0). (b) 0.5 mM Trp-Trp dipeptides in PSSS-templated Ag NPs (pH 6.0). (B) Steady state fluorescence emission spectra (a) 0.5 mM Tryptophan solution. (b) 0.5 mM Tryptophan in PSSS-templated Ag NPs.
To further understand the quenching mechanism, the fluorescence lifetime of Trp-Trp dipeptides in silver colloids were measured by time correlated single photon counting, as shown in Table 1 . Trp-Trp dipeptides in phosphate buffer (pH 6.0, Samples 1) have three lifetimes: long (2.1 ns), intermediate (1.0 ns), and short (0.4 ns). After the addition of PSSS-templated silver Ag NPs, the long lifetime of Trp-Trp dipeptides increases by almost 0.5 ns (Table 1, Samples 2).
Table 1. Trp-Trp dipeptides lifetimes, pH 6.0, room temperature
In the Trp-Trp dipeptides/metal silver system, Trp-Trp dipeptides adsorbed onto the silver surface can serve as ligands, and there is charge transfer between the indole ring and the silver surface. The 0.5 ns increased lifetime is related to this charge transfer. Figure 6 shows fluorescence decays at different emission wavelengths, which are independent of the emission wavelength, and the increased 0.5 ns lifetime is relatively constant. Thus, the quenching mechanism must be dynamic. The quenching mechanism of Trp-Trp dipeptides on silver surface can provide useful information to study adsorption behaviors of analogous peptides and proteins in biological science.
4. Conclusions
We investigated the spectral features of Trp-Trp dipeptides on PSSS-templated Ag NPs using UV-Vis, SERS, and fluorescence spectroscopy. The PSSS-templated Ag NPs wrapped aromatic moieties have high adsorption affinity to the indole ring of Trp-Trp dipeptides via π-π stacking interaction mechanism. A new SPA band of Ag NPs showed that the indole rings of Trp-Trp dipeptides can bind to nearby Ag NPs to form the dimer. SERS spectra confirmed that the adsorption sites on silver surface are indole rings. Charge transfer between the indole ring and Ag NPs, attenuated the fluorescence intensity of Trp-Trp dipeptides by almost 30%. The lifetime of Trp-Trp dipeptides increased by 0.5 ns, which was independent of the emission wavelength. The spectral features of Trp-Trp dipeptides on PSSS–templated Ag NPs provide useful information to study analogous peptides, proteins, and enzymes in life science.
Acknowledgments
Parts of this work were supported by the National Natural Science Foundation of China (No. 61378091, 11204226); National Basic Research Program of China (Grant No. 2015CB352005); Guangdong Natural Science Foundation (2014A030312008); Shenzhen Basic Research Project (ZDSYS20140430164957663 /KQCX20140509172719305); the Training Plan of Guangdong Province Outstanding Young Teachers in Higher Education Institutions (Yq2013142), and the Science and Technology Innovation Project of Guangdong Province (2013KJCX0158).
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