Article pubs.acs.org/JPCC
Energy Transfer from C‑Phycocyanin to Single-Walled Carbon Nanotubes Karim El Hadj, Patricia Bertoncini,* and Olivier Chauvet Institut des Matériaux Jean Rouxel, Nantes Université, CNRS 2 rue de la Houssinière, BP 32229, 44322 Nantes, France ABSTRACT: The energy transfer from C-phycocyanin (C-PC) to single-walled carbon nanotubes (SWNTs) in SWNT/C-PC hybrids is investigated using optical absorption and fluorescence spectroscopy. A suspension-dialysis method is used to assemble the SWNTs and C-PC proteins. The proteins cover partially the SWNT length as observed on atomic force microscopy images and transmission electron microscopy micrographs. Photoluminescence measurements show a strong enhancement of emission intensity of several SWNT species, while a strong decrease of the C-PC emission occurs in the presence of SWNTs. Our data suggest that a nonradiative resonance energy transfer takes place after the excitation of the C-PC with a high efficiency.
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focuses on the fluorescence properties of SWNT/C-PC hybrids and shows that a resonant energy transfer occurs from C-PC to SWNTs in the hybrids.
INTRODUCTION Single-walled carbon nanotube (SWNT) fluorescence properties are very attractive for their use in biosensing, biomedical imaging, and optoelectronics.1−3 The SWNT emission takes place in the near-infrared range, so at wavelengths that do not interfere with biological tissue absorption, it is highly stable and very sensitive to changes in the close environment of the nanotubes. To get access to the fluorescence properties, SWNTs have to be functionalized noncovalently. This can be done using light-absorbing and light-emitting molecules, such as organic dyes,4−7 semiconducting nanoparticles/quantum dots,8 and conjugated polymers.9,10 These systems exhibit energy transfers that can be useful for many applications. Recently, we demonstrated that radiative and nonradiative energy transfers can also occur in SWNT/bacteriorhodopsin hybrids.11 In this work, we choose to work with C-phycocyanin (C-PC). C-PC is a phycobiliprotein present in cyanobacteria which possesses light-harvesting properties.12 It absorbs light between 615 and 620 nm and then provides a highly efficient exciton migration until the energy arrives at a photochemical reaction center through a tunneling mechanism; it strongly emits light around 647 nm; and it is able to accept or transfer energy to other phycocyanins, like phycoerythrin or allophycocyanin. C-PC is composed of two dissimilar α and β protein subunits of 19 500 and 21 500 Da, and it has some hydrophobic residues on its surface.12 Phycobiliproteins are extensively commercialized for fluorescent applications in clinical and immunological experiments and used as a colorant.13 Recently, Bora et al. showed that a hematite−phycocyanin integrated system can work as a photoanode for photoelectrochemical applications.14 In this work, the C-phycocyanin proteins are noncovalently immobilized onto the sidewall of SWNTs with the help of a sodium cholate suspension dialysis method.15 Our study © 2014 American Chemical Society
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EXPERIMENTAL SECTION Materials. HipCO SWNTs were obtained from Carbon Nanotechnologies Inc. Co. (Houston, TX) and used without any further purification. Sodium cholate (SC) and HEPES (4(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) have been purchased from Sigma-Aldrich, and C-phycocyanin (C-PC) suspension was purchased from AnaSpec. Before use, the C-PC suspension was centrifuged at 10 000g for 30 min. The supernatant was then discarded and the pellet resuspended into a 5 mM HEPES buffer at pH 7.5. Manipulation of C-PC was done away from light. Preparation of SWNT/SC Stock Dispersion. SWNTs were added to a 40 mM aqueous solution of SC with a concentration of 0.5 mg/mL and dispersed by ultrasounds using first a cup horn during 15 min and then a bath for 3 h. Aggregates of nanotubes were eliminated by centrifugation at 7200g for 2 h. Preparation of SWNT/C-PC Dispersion. An amount of 5 μL of a C-PC solution with a concentration of 0.4 mg/mL was added to 1 mL of a SWNT/SC suspension. The mixture was dialyzed for 5 h against 1 L of HEPES buffer at a concentration of 5 mM and at pH 7.5 using a 10 kDa dialysis membrane to remove the excess of SC molecules. Then the dispersion was transferred to a 100 kDa dialysis membrane for 1 h to remove unabsorbed monomeric and dimeric C-PC proteins. Finally, the dispersion was centrifuged for 30 min at 7200g, and the Received: November 27, 2013 Revised: February 24, 2014 Published: March 4, 2014 5159
dx.doi.org/10.1021/jp411653s | J. Phys. Chem. C 2014, 118, 5159−5163
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SC suspensions were recorded immediately after sonication and before centrifugation. The absorbance of these suspensions reflects 100% of the initial known SWNT amount (0.5, 0.25, 0.125, 0.02, and 0.01 mg/mL SWNTs). The optical density (OD) of these suspensions was measured at a wavelength of 891 nm (where SWNTs do not present interband transitions) and used for plotting a calibration line giving the OD as a function of the SWNT concentration. We found a calibration value for SWNTs of 0.29 mg/mL for absorption of 1 OD at 891 nm. Then the optical density of the SWNT suspensions containing the proteins was measured, and we found a final concentration of SWNTs of about 0.17 mg/mL. The concentration of C-PC was calculated by subtracting contributions of SWNTs to absorbance at 615 nm and using an extinction coefficient of 1.9 × 106 M−1 cm−1 and was about 16 nM. Figure 2A shows a typical 3D topographical AFM image of a rinsed film supernatant containing SWNTs and C-PC. The elongated structure can be attributed to a SWNT with a height of 0.7 nm. Proteins can be seen on top of the mica surface and onto the SWNT. For the sake of comparison, a 3D topographical AFM image of a rinsed film supernatant containing the initial SWNT/SC suspension is also shown in Figure 2C. The height profiles recorded along the SWNT present in Figure 2A and 2C are presented in Figure 2B and 2D, respectively. The height profiles of Figure 2D and 2B exhibit heights ranging from 0.5 to 3.0 nm and 0.7 to 8.7 nm, respectively. The C-PC proteins are partially covering the SWNT forming a hybrid. This is the case for all AFM images that we recorded. In fact, by measuring the naked and covered length of 15 SWNT/C-PC hybrids on several AFM images, we found that the proteins cover approximately 60% of the nanotube length with values ranging from 50 to 70%. The majority of the proteins has heights in the range of 2−4 nm, and a few of them can have heights as high as 7.6 nm. For comparison, protein height values were measured on AFM images recorded on C-PC proteins originating from a C-PC suspension in 5 mM HEPES buffer. In addition to the heights mentioned before, height values lower than 2 nm, in particular around 1.3 nm, were found. These values may correspond to unbound monomeric (41 kDa) or dimeric (82 kDa) C-PC which are expected to be removed during the second dialysis step at 100 kDa. The lengths of the SWNTs are quite short, ranging from 100 to 1900 nm (average length is (500 ± 20) nm). This is likely due to the sonication step used to disperse the carbon nanotubes. A typical TEM micrograph is shown in Figure 3. The straight lines correspond to the sidewall of a nanotube which has a diameter of 1.3 nm. Naked and covered areas can be seen on the nanotube as previously observed by AFM. The whole diameter of the areas which are covered by proteins is about 3− 5 nm, in agreement with AFM observations. The NIR emissions from various SWNT species were recorded with excitation wavelengths ranging from 550 to 700 nm. Figure 4 shows the photoluminescence excitation/emission maps for SWNTs embedded in sodium cholate micelles for the sake of comparison (A) and for SWNT/C-PC hybrids (B). On the basis of the emission wavelength from first van Hove singularity transition (E11) and the excitation wavelength from their second van Hove singularity transitions (E22),18 five major SWNT chiral species, (6,5), (8,4), (8,3), (7,5), and (7,6), are identified in Figure 4 as solid diamonds. Additional fluorescence spots appear for SWNT/C-PC hybrids. Emission
supernatant was preserved for further analysis. This preparation was done away from light. Spectroscopic Measurements. The dispersions were optically characterized by UV−vis−NIR absorption using a CARY 5G spectrometer. Photoluminescence maps and luminescence spectra were recorded using a Jobin-Yvon Fluorolog spectrometer equipped with an UV−vis Xenon lamp. An InGaAs IR detector (427C-AU Horiba) and a CCD camera were used for the signal detection. Atomic Force Microscopy (AFM). A suspension of SWNT/C-PC dispersion was diluted twice. Then 10 μL was deposited onto a freshly cleaved mica surface for 10 min before rinsing with water and drying under N2 flux. The topography of the formed assemblies was characterized in air using a Nanowizard II AFM (JPK Instruments, Berlin, Germany) operating in the intermittent contact mode. Tips used were made of silicon (PPP-NCHR, Nanosensors). Transmission Electron Microscopy (TEM). Samples were prepared by dipping a grid covered by a holey carbon film into 20 μL of a SWNT/C-PC suspension. Micrographs were recorded using a Hitachi HF-2000 equipped with a field emission gun and operating at 100 kV.
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RESULTS AND DISCUSSION The supernatants containing the SWNTs dispersed by sodium cholate and those containing the SWNTs and the C-PC are gray colored. Their UV−visible absorption spectra are shown in Figure 1. An UV−visible absorption spectrum of C-PC is also
Figure 1. UV−visible absorption spectra of supernatants between 220 and 1350 nm of well-dispersed SWNTs by sodium cholate (black line) and of SWNT/C-PC hybrids in buffer solution (gray line). In addition, an UV−visible spectrum of C-PC between 400 and 800 nm is also shown in the inset, and the signal is normalized at 616 nm.
shown in the inset of Figure 1, exhibiting a maximum absorption band at ∼615 nm. Partly resolved absorption peaks can be seen between 400 and 1350 nm and are due to S11 and S22 optical transitions from well-suspended SWNTs.16 For supernatants containing also the proteins, an increase of the absorption signal is visible at wavelengths around 615 nm that is due to protein absorption. The optical absorption spectroscopy measurements were used to determine the SWNT concentration in the suspensions following the procedure described by Haggenmueller et al.17 and the protein concentration. Briefly, absorbance spectra of several SWNT/ 5160
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Figure 2. (A) AFM 3D image showing an individual SWNT/C-PC hybrid deposited onto a mica surface. (B) Height profile recorded along the green line on image A. (C) AFM 3D image showing an individual SWNT individualized by sodium cholate molecules. (D) Height profile recorded along the green line on image C.
transitions of the SWNTs. The position and excitation profile of the emission do, however, closely match the optical absorbance maximum of the protein, which occurs around 620 nm. Moreover a spectral overlap exists between the emission of the C-PC and the absorption of several SWNT chiral species for wavelengths that are in the range of 600−700 nm. This can be clearly visualized on Figure 5 which shows the absorption spectrum of SWNTs (black curve) and the protein photoluminescence spectrum when excited at 617 nm (gray curve). Photoluminescence emission (PLE) spectra of supernatants containing the SWNT/C-PC hybrids, and the SWNTs embedded in SC micelles are presented in Figure 6 when
Figure 3. Transmission electron micrographs taken at 100 kV showing an individual single-walled carbon nanotube coated with C-PC proteins.
occurs from nanotube species present when excitation wavelengths are in the range 600−620 nm. These spectral areas do not correspond to any known active electronic
Figure 4. Steady-state fluorescence maps of a dispersion of SWNTs obtained using sodium cholate (A) and a dispersion containing SWNT/C-PC hybrids (B). White diamonds indicate five carbon nanotubes species. 5161
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Figure 7. PLE spectra of free C-PC (black curve) and of SWNT/CPC hybrids (gray curve) excited at 616 nm.
Figure 5. Absorption spectrum of well-dispersed SWNTs using sodium cholate, normalized at 400 nm (black line). PLE spectrum of free C-PC (gray line) detected for an excitation wavelength of 616 nm with a laser power of less than 5 mW.
where ID and IDA are the fluorescence of the donor in the absence and presence of an acceptor, respectively. The efficiency in our system is estimated to be around 0.89.
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excited at a wavelength of 635 nm (Figure 6A) and at a wavelength of 670 nm (Figure 6B). Five SWNT species are identified with their chiral numbers (n,m) for emission wavelengths ranging from 825 to 1300 nm. The intensity of the PLE spectrum of SWNT/C-PC hybrids excited at 635 nm (gray curve in Figure 6A) is greatly enhanced compared to the SWNTs embedded in sodium cholate micelles (black curve in Figure 6A). This is not the case when the SWNT/C-PC hybrids are excited at 670 nm, a wavelength that is outside the spectral overlap (Figure 6B). Only small modulations in intensity can be seen. Figure 7 presents the fluorescence emissions of the proteins for free C-PC in buffer (black curve) and for SWNT/C-PC hybrids (gray curve). These emissions arise from the same amount of proteins. In the case of the hybrids, a decrease in intensity of about a factor of ten is measured. These observations altogether are in favor of a resonance energy transfer (RET). Upon red light excitation, the energy is transferred resonantly to SWNTs, and the C-PCs return to their ground state without emitting visible light. The efficiency (E) of a RET process can be derived experimentally from the quenching of the donor fluorescence intensity, according to19 I E = 1 − DA ID
CONCLUSIONS This study provides evidence that the C-phycocyanin proteins can be immobilized on the sidewall of single-walled carbon nanotubes. The carbon nanotube is partially covered by proteins as observed by electronic and atomic force microscopy. By carefully studying the fluorescence emission of the protein in the presence and in absence of SWNTs and the photoluminescence of the SWNTs in the near-infrared region, we demonstrate that a resonance energy transfer occurs from the light-harvesting proteins to the SWNTs after protein photoexcitation. The proteins keep their activity during the absorption step. Successful association of these two molecules is of great interest regarding possible optoelectronic applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS C-PC, C-phycocyanin; SWNTs, single-walled carbon nanotubes; SC, sodium cholate; HEPES, 4-(2-hydroxyethyl)
Figure 6. PLE spectra of a dispersion of SWNT/SC (black curve) and SWNT/C-PC hybrids (gray curve) excited at 635 nm (A) and at 670 nm (B). 5162
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piperazine-1-ethanesulfonic acid; UV−vis−NIR, ultraviolet− visible−near-infrared; AFM, atomic force microscopy; TEM, transmission electron microscopy; PLE, photoluminescence emission; RET, resonance energy transfer
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