Article pubs.acs.org/JPCC
Distance-Dependent Excited-State Electron Transfer from Tryptophan to Gold Nanoparticles through Polyproline Helices Ying-Chen Lai,† Chih-Ying Lin,† Meng-Ru Chung,† Pei-Yu Hung,† Jia-Cherng Horng,*,†,‡ I-Chia Chen,†,‡ and Li-Kang Chu*,†,‡ †
Department of Chemistry and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan S Supporting Information *
ABSTRACT: A series of polypeptides (CAPnWNs, where n = 3, 6, 10, and 13), mainly composed of consecutive prolines, were covalently anchored onto the surfaces of gold nanoparticles through the terminal cysteine to form Au−S bonds. A tryptophan near the C-terminus of the peptide exposed to water serves as a fluorophore. By changing the number of prolines, the distance between the tryptophan and the gold nanoparticle can be systematically adjusted within the range of 5 nm. The kinetics of the tryptophan fluorescence in pure CAPnWNs at 355−365 nm upon 266 nm excitation is insignificantly accelerated in comparison with that of pure tryptophan. As CAPnWNs are attached onto ca. 13 nm gold nanoparticles, the fluorescence kinetics of tryptophan is drastically accelerated as its distance to the gold nanoparticle surface is shortened. Accounting for the nanosurface energy transfer (NSET) and electron transfer process from the excited state of tryptophan to the gold nanoparticle, an apparent decay factor β attributed to electron transfer is derived as 0.11 Å−1, referring to a long-distance electron transfer pathway. An additional rate coefficient, attributed to the tryptophan−tryptophan intermolecular energy transfer at a short distance, was also taken into consideration and suggested the close occupancy of CAPnWNs on the nanoparticle surface. Our results further suggested that polyprolines are capable of serving as molecular rulers and molecular wires in conjugating with nanomaterials. component is of minor contribution (99%) was added as a reducing agent. After 40 min reaction at 80 °C, the color of the mixture changed from light yellow to bright red, indicating the complete reduction of Au3+. The colloidal solution was then cooled and stored at room temperature for further experiments. According to the TEM image (JSM-7000F, JEOL), the average size of the as-prepared particles was about 14.2 ± 1.0 nm (Figure S4b). Anchoring CAPnWNs on Gold Nanoparticles (AuNPCAPnWNs). AuNP-CAPnWNs were prepared by mixing the as-prepared gold nanoparticle solution with an excess of CAPnWNs in a molar ratio of 105. The AuNP and CAPnWN concentrations were determined using steady-state absorption spectroscopy. The extinction coefficients of 14 nm AuNP at the SPR band and the single tryptophan in CAPnWNs at 280 nm were 3 × 108 M−1 cm−1 (ref 42) and 5880 M−1 cm−1 (ref 43), respectively. The pH of the mixture was then adjusted to 9 with 0.1 M NaOH in drops and stirred overnight at room temperature. The mixture turned dark red in color when the reaction completed. In order to reduce the interference in the fluorescence measurement due to unanchored polyprolines, the mixture was centrifuged at 1150g for 30 min, and the concentrate was redistributed in fresh deionized water. This procedure was performed four times, and the final precipitates of the AuNP-CAPnWNs were dispersed in 0.4 mL of deionized water for further measurements. In order to confirm the removal of unreacted polyprolines, the supernatants after the third centrifugation were characterized using infrared and fluorescence spectroscopy. The successful attachment of polyprolines onto AuNP surfaces was also confirmed by the same spectroscopic methods.
precisely control the distance between a nanoparticle and a fluorophore to within 5 nm is challenging. In this work, we inspected the fluorescence kinetics of tryptophan in proximity of 14 nm gold nanoparticles separated by polyprolines of different lengths within 5 nm (CAPnWNs, n = 3, 6, 10, and 13, as shown in Figure 1). Lévy et al. have successfully anchored polypeptides onto gold nanoparticles through Au−S bonding by modifying the terminus with cysteine.39 Similarly, we modified the surface of the gold nanoparticles with polyprolines of different lengths. The waterexposed moiety contains a tryptophan to serve as the fluorophore and is terminated with an asparagine to increase the solubility. We found that the lifetime of the tryptophan fluorescence becomes shortened as the distance between the tryptophan and the gold nanoparticle decreases. The electron transfer process was taken into consideration to explain the distance-dependent fluorescence kinetics of tryptophan. The goal of this work is to provide the fundamentals for utilizing polyprolines as precise molecular rulers and molecular wires in the conjugation of biological molecules and gold nanoparticles.
2. MATERIALS AND METHODS Materials Preparation. Synthesis of Polypeptides CAPnWNs. Tryptophan-containing peptides of different numbers of prolines, CAPnWNs (n = 3, 6, 10, and 13), were synthesized on the 0.2 mmol scale for CAP3WNs and the 0.1 mmol scale for other peptides using solid-phase peptide synthesis (SPPS).40 9-Fluorenylmethyloxycarbonyl (Fmoc)protected amino acids were used in the synthesis. The piperidine-assisted deprotection and HBTU-activated coupling cycles were carried out on a microwave peptide synthesizer (CEM, Discover SPS), and the N-terminus of each peptide was acetylated by acetic anhydride at the final step. Acetylation of the N-terminus of a peptide was to avoid charge repulsions under the experimental condition. The use of Rink amide resin generated an amidated C-terminus upon cleavage. The cleavage of peptides from Rink amide resin was carried out with 94% trifluoroacetic acid/1% triisopropylsilane/2.5% ethanedithiol/ 2.5% H2O (v/v). All crude peptides were purified by reversed phase HPLC with a Vydac semipreparative C18-column and gradient elution solvents of H2O/acetonitrile containing 0.1% (w/v) trifluoroacetic acid. The purity of purified peptides was greater than 95% according to HPLC analysis, and the B
DOI: 10.1021/acs.jpcc.6b12640 J. Phys. Chem. C XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry C Steady-State Spectroscopy. Steady-state ultraviolet− visible (UV−vis) absorption spectra were recorded with a spectrometer (USB4000-UV−vis, Ocean Optics) using a 1 cm quartz cuvette. A continuous-scan Fourier-transform infrared spectrometer (Vertex 80, Bruker), coupled with a multipass attenuated total reflection (ATR) from BioATR Cell II (Bruker), was utilized to collect the infrared (IR) absorption spectra of the samples dried in the sample compartment of the ATR with a spectral resolution of 1 cm−1; each spectrum was averaged from 50 scans. The fluorescence (FL) spectra were collected using an F-7000 Hitachi spectrophotometer. The emission contours were recorded from 280 to 500 nm upon excitation at 266 nm. Circular dichroism (CD) measurements were conducted on an Aviv-410 CD spectrometer with a 1 mm path length quartz cuvette. The peptide was dissolved in water with a concentration of 70 μM. The far-UV CD measurements were recorded at 25 °C (Figure S2) to confirm the configuration of the helical structures. Time-Correlated Single Photon Counting (TCSPC). A Maitai femtosecond laser (Spectra-Physics, at 80 MHz repetition rate) provided 800 nm photons. A set of BBO crystals (Type I, Eksma Optics) was employed to generate the third harmonic wavelength at 266 nm. A trigger signal from an avalanche photodiode (PicoQuant, TDA-200) was sent to a Hydra Harp 400 (Picoquant) for data recording. An MCPPMT (Hamamatsu, R3809, U-57) was used to collect the fluorescence, which was further amplified using a preamplifier (Hamamatsu C5594). A filter was mounted in front of the MCP-PMT to define the detection wavelengths in the 355− 365 nm range. The data were acquired for 30 min for a better ratio of signal-to-noise. The instrumental response was about 30 ps. Estimations of Lengths of CAPnWNs. The structure of CAP6WN in water was derived by density functional theory with Hartree−Fock method using basis set of 6-31+g(d).44 The predicted lengths of cysteine-alanine moiety at the N-terminus and distance between the polyproline and tryptophan at the Cterminus were used for determining the other CAPnWNs with a deviation of 2 and 1 Å to include the possible uncertainty. The lengths of repeating proline units are determined using a reported value of 3.1 Å per proline.3,7
Figure 2. (a) Normalized steady-state extinction spectra, with respect to the plasmonic band, of the ca. 12−14 nm AuNP and polyprolinecapped gold nanoparticles, AuNP-CAP3WNs, AuNP-CAP6WNs, AuNP-CAP10WNs, and AuNP-CAP13WNs. (b−f) Electron microscopy images of the nanoparticles mentioned above, with the histograms of the size distributions in the insets. The samples were redistributed from the purified precipitates to exclude the unanchored PP. The spectra and TEM images of as-prepared samples are supplemented in Figure S4.
Figure 2a, indicated an increase in the size of AuNPs from 17 to 45 nm. However, the electron microscope images, shown in Figures 2b−f, do not show significant changes in the diameters of the AuNP-CAPnWNs after purification by centrifugation. The observations suggest that the spectral shift was due to the intrinsic properties of the AuNP-CAPnWNs; the attachment of CAPnWNs altered the dielectric properties of the proximity of gold nanoparticles. Infrared Spectra. The infrared absorption spectra of the pure CAPnWNs are shown in gray traces in Figure 3b−e. The main vibrational features are the amide I band (the CO stretching mode of the peptide bond at 1634 cm−1), amide II band (C−N−H deformation at 1531 cm−1), and amide A band (N−H stretching mode at 3300 cm−1).47 The presence of prolines in CAPnWNs displays a stronger absorbance at 1432 cm−1, which is attributed to the C−H deformation of the proline side chain.48 These absorption features appear on the AuNP-CAPnWN samples (colored traces in Figure 3b−e) after substituting the citrate features (gray trace in Figure 3a), suggesting the successful attachment of the CAPnWNs onto the gold nanoparticles. Based on the results of Johnston and Krimm, the wavenumber of the CO stretch of poly(Lproline) shifts from 1619 to 1643 cm−1 as the sample is dried.49 They also demonstrated that the aggregates could form by excluding water and that the CO wavenumber corresponds to the non-hydrogen-bonded groups in solid.49 Therefore, a minute blue-shift of amide I in each AuNP-CAPnWN sample in
3. RESULTS AND DISCUSSION The attachment of CAPnWNs to gold nanoparticle surfaces was confirmed using multipass ATR infrared absorption, steadystate absorption, and fluorescence. The lifetimes of the tryptophan fluorescence were recorded with TCSPC. The observed difference in the fluorescence kinetics will be discussed in terms of energy transfer and electron transfer processes. Steady-State Spectroscopic Characterization of the AuNP-CAPnWNs. UV−Vis Extinction Spectra. The extinction spectra of AuNP-CAPnWNs after centrifugation purification (Figure 2a) exhibited a slight red-shift in the surface plasmonic resonance (SPR) bands toward ca. 530 nm in comparison with the as-prepared gold nanoparticles characterized at 521 nm (Figure S4a). Generally, the red-shift of the SPR band indicates an increase in particle size if the surface dielectric properties of the gold nanoparticles are unchanged.45 On the basis of the relationship of the wavelength at absorption maximum and the diameter of gold nanoparticles
