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OPTICS LETTERS / Vol. 40, No. 7 / April 1, 2015
Two-photon-induced Förster resonance energy transfer in a hybrid material engineered from quantum dots and bacteriorhodopsin Victor Krivenkov,1,2,* Pavel Samokhvalov,1 Daria Solovyeva,1 Regina Bilan,1 Alexander Chistyakov,1,2 and Igor Nabiev1,3,4 1
Laboratory of Nano-Bioengineering of National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation 2 Department of Micro- and Nanophysics of National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation 3 Laboratoire de Recherche en Nanosciences, LRN-EA4682, Université de Reims Champagne-Ardenne, 51 rue Cognacq Jay, 51100 Reims, France 4 e-mail: [email protected] *Corresponding author: [email protected] Received January 16, 2015; revised February 25, 2015; accepted March 2, 2015; posted March 3, 2015 (Doc. ID 230560); published March 27, 2015 Energy transfer from nanostructures to biological supramolecular photosystems is an important fundamental issue related to the possible influence of nanoobjects on biological functions. We demonstrate here two-photon-induced Förster resonance energy transfer (FRET) from fluorescent CdSe/ZnS quantum dots (QDs) to the photosensitive protein bacteriorhodopsin (bR) in a QD–bR hybrid material. The two-photon absorption cross section of QDs has been found to be about two orders of magnitude larger than that of bR. Therefore, highly selective two-photon excitation of QDs in QD–bR complexes is possible. Moreover, the efficiency of FRET from QDs to bR is sufficient to initiate bR photoconversion through two-photon excitation of QDs in the infrared spectral region. The data demonstrate that the effective spectral range in which the bR biological function is excited can be extended beyond the band where the protein itself utilizes light energy, which could open new ways to use this promising biotechnological material. © 2015 Optical Society of America OCIS codes: (160.1435) Biomaterials; (160.4236) Nanomaterials; (160.5335) Photosensitive materials; (260.2160) Energy transfer; (020.4180) Multiphoton processes. http://dx.doi.org/10.1364/OL.40.001440
The development of novel nano-hybrid structures for light-harvesting, photovoltaic, and light-emitting devices [1] is a promising field of application of fluorescent semiconductor nanocrystals or quantum dots (QDs) [2]. Energy transfer from and between QDs plays an important role in nano–bio hybrid structures based on QDs and photosensitive biomolecules [3]. Bacteriorhodopsin (bR), a unique photosensitive protein, is known to express its photovoltaic properties upon excitation with visible light [4]. bR exposed to radiation of the solar spectrum utilizes as little as 0.1%–0.5% of the solar energy [5]. It has been shown that the use of QDs, which absorb light in a wide range, including the ultraviolet (UV) region strongly increases the efficiency of bR photovoltaic bioconversion because of single-photon Förster resonance energy transfer (FRET) from QDs to the protein [6,7]. However, single-photon excitation in QD–bR complexes is nonselective, because irradiation in the visible spectral region excites both components of the hybrid system (bR and QDs). The two-photon absorption cross section (TPACS) of QDs [8,9] is two orders of magnitude larger than maximal TPACS of bR (290 GM) [10]. This means that two-photon excitation of a QD–bR hybrid material may, first, ensure a high selectivity of QD excitation in QD–bR complexes and, second, make it possible to initiate the bR biological function in the infrared region, which would open new ways to use this material. The purpose of this study was to prepare and characterize tight self-assembled complexes of bR and QDs and to analyze the efficiencies of single-photon-excited and 0146-9592/15/071440-04$15.00/0
two-photon-excited FRETs between them in this hybrid material. The main goal of the study was to estimate the possibility of initiation of the bR biological function through two-photon-induced FRETs from fluorescent CdSe/ZnS QDs to bR in the QD–bR complexes. The core/shell CdSe/ZnS QDs for our experiments were synthesized by the hot injection method [11] and then made water soluble by replacing the surface ligand TOPO with the SH-polyethyleneglycol-OH (SH-PEG-OH) polymer as described earlier [12]. The fluorescence maxima of these QDs were at 570 nm and coincide with the absorption maxima of bR (Fig. 1). The QD QY was 14%, and the overlap integral for the QD–bR pair was 6.1 · 1015 M−1 cm−1 nm4 . We used bR in its native purple
Fig. 1. Normalized QD fluorescence and absorption spectra, and the normalized PM absorbance spectrum. © 2015 Optical Society of America
April 1, 2015 / Vol. 40, No. 7 / OPTICS LETTERS
membranes (PMs) extracted from the bacteria H. salinarum by the standard procedure [4]. White membranes (WMs) were prepared by carefully extracting the only photosensitive chromophore (retinal, the FRET acceptor) from the PMs [7]. Two-photon excitation of the samples was performed by a femtosecond laser system at a wavelength of 790 nm, pulse energy of 5 nJ, and pulse duration of 300 fs. The laser beam was focused with a 10 cm focal length lens. One-photon excitation was performed by means of the same system at a wavelength of 395 nm, pulse energy of 0.15 nJ, and pulse duration of 300 fs. To eliminate the influence of the inner filter effect on the fluorescence intensity on an increase in the acceptor concentration in our samples, the data were corrected using the correction factor calculated as described in [13]. The TPACS of QDs was calculated on the basis of comparative measurements of two-photon-induced fluorescence of nanocrystals and reference organic dyes with known TPACS as described in [14]. Fluorescein and Rhodamine 6G were used as reference dyes for TPACS determination [15]. The concentrations of QDs in solutions were estimated using the extinction coefficients in the first exciton maxima for QDs of different diameters determined in [16]. Prior to experiments on two-photon excitation of QD– PM systems, we had to test whether the nano–bio hybrid complexes of QDs and PMs were really formed and prove the possibility of one-photon FRET in our system. To do this, we monitored the FRET efficiency parameter in water mixtures of bR and QDs at different bR-to-QD molar ratios. The FRET efficiency on the experiment was calculated by comparing the fluorescence quantum yield (QY) of QDs in water with the QY of QDs mixed with a suspension of PMs. The equation for the FRET efficiency–E was obtained by substituting QY parameters for fluorescence lifetimes in Equation 13.14 from [17]: E 1 − φ∕φ0 , where φ is the QY of the QD in the presence of the PM, and φ0 is the QY of the QD in the absence of the PM. It is worth mentioning that the degradation of QY of QDs in QD–bR complexes may be related not only to the FRET from QD to bR, but also to other quenching mechanisms. To test whether FRET was the main QD quenching mechanism in our samples, we have compared the time courses of QD fluorescence quenching in QD–PM complexes and the complexes of QDs and WMs. WMs may serve as an excellent negative control for the evaluation of FRET efficiency in hybrid materials. WMs and PMs have exactly the same physical, chemical, and structural properties (lipid composition, surface charge, fluidity, rigidity, etc.). Hence, they self-assemble and form the complexes of exactly the same structure and morphology with QDs. The only difference between the QD–PM and QD–WM complexes is that FRET is impossible in the QD–WM complexes because of the absence of the FRET acceptor (retinal). To test whether the FRET occurred in QD–PM complexes upon one-photon excitation, we gradually added aliquots of a PM suspension to a water solution of QDs with a molar concentration of 2 · 10−6 M and measured the average fluorescence lifetimes (AFLs) and integrated fluorescence intensities of the resultant solutions (Fig. 2). An increase in the protein concentration in the QD–PM
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Fig. 2. Dependences of (a) fluorescence intensity and (b) average fluorescence lifetimes on the bR-to-QD molar ratio in the complexes of QDs with PMs upon the excitation at 395 nm. The data obtained for the negative control (complexes of QDs with WMs) are also shown.
system caused a pronounced decrease in both fluorescence intensity and AFL. In the QD–WM system, this decrease was much smaller. The difference between the samples containing PMs and WMs can be explained by assuming a zero overlap integral for the QD–WM system, in which the acceptor is absent. These data clearly confirm that the decrease in the fluorescence intensity and AFL observed in the QD–PM samples was determined by the FRET between QDs and bR. As we have speculated in our previous study [7], the self-assembly of QDs on the surface of a PM or WM may be determined by the electrostatic interaction of negatively charged QD surface groups and positively charged amino acid residues of bR located on the PM surface. To determine whether the FRET in our samples was because of the formation of tight QD–PM complexes or merely short distances from the free-floating donor particles (QDs) to the acceptor in the solution, we measured the dependence of the FRET efficiency on the concentration of QDs in the solution and compared it with the theoretical predictions for a free-particle system. The distance between the particle—r is inversely proportional to the cube root of the concentration—C. Thus, the distance changes from r 0 to r when the concentration changes from C 0 to C, by the following equation: r r 0 C 0 ∕C1∕3 :
(1)
Thus, regarding the free-floating particle system, by dilution of the sample, we would vary the concentrations and, consequently, the distance between the donor and acceptor in the aqueous mixtures of QDs and PM without changing their molar ratio. The FRET efficiency (E) can be expressed from [17]: E R60 R60 r 6 −1 ;
(2)
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where R0 is the Forster radius. Using Eqs. (1) and (2), we can estimate the dependence of the E on the C by the following equation: E R60 R60 r 60 C 0 ∕C2 −1 ;
(3)
where C 0 is the solution concentration when the distance between particles is r 0 and the FRET efficiency is known and equal to E 0 . R0 does not change with changing concentration; hence, it can be expressed in terms of r 0 and E0 using Eq. (2): R0 r 0 E0 1 − E 0 −1 1∕6 :
(4)
Thus, by substituting R0 from Eq. (4) into Eq. (3), we obtain the final expression for the dependence of the FRET efficiency on the concentration of the solution: 2 −1 E 1 E −1 0 1 − E 0 C 0 ∕C :
(5)
Next, we measured the experimental dependence of the FRET efficiency on the concentration of solution, to compare with the theoretical dependence and validate our assumption that the system is not the set of free-floating particles, but it is an aqueous solution of the QD-bR complexes. To obtain the experimental dependence of the FRET efficiency from the solution concentration, we used an aqueous mixture of QDs and PMs with an initial FRET efficiency (E 0 ) of 0.4, a bR-to-QD molar ratio of 4, and a QD concentration of 2 · 10−7 M. The stock solution was diluted sequentially 16 times, and the QY was measured at each dilution step. Figure 3 shows the theoretical and experimental dependences of the FRET efficiency on the concentration of the solution. It is clear that the FRET efficiency is much higher than could be expected from the model of free-floating particles at every experimental point. The observed decrease in the FRET efficiency (from 0.4 to 0.25 after 16-fold dilution of the sample) can be explained by assuming that the complexes were formed upon adsorption of QDs onto PMs, and there was a dynamic equilibrium between the desorbed QDs and QD–PM complexes in the solutions studied. Although efficient one-photon-induced FRET in QD– bR hybrid materials has been demonstrated earlier [1,6,7], two-photon-induced FRET from a nanomaterial to a photosensitive biological system has never been observed before to the best of our knowledge. The possibility to induce or enhance a biological function through excitation in an optical region wider than the spectral
Fig. 3. Experimental FRET efficiency upon dilution of the QD–bR sample and the theoretically predicted FRET efficiency for a free-floating particle system Eq. (5).
range where natural biosystems utilize light energy may open new prospects for biophotonic applications and the development of bioinspired devices. Therefore, we have further analyzed the possibility of efficient two-photoninduced FRETs in tight QD–PM complexes developed, and structurally and optically characterized above. First, we calculated the TPACS of our QDs and found it to be two orders of magnitude higher than that of bR. The measured value of the QD TPACS at an excitation wavelength of 790 nm was about 20,000 GM. The FRET in the QD–PM system upon two-photon excitation was induced in a manner similar to that of one-photonexcitation described above. The dependences of the fluorescence intensity and AFL on the bR-to-QD molar ratio are shown in Fig. 4. It can be seen that an increase in the PM concentration leads to a decrease in the fluorescence intensity and AFL in the QD–PM system. As in the case of one-photon excitation, this decrease was much smaller in the QD–WM system. These data show that FRET occurs in QD–PM hybrid complexes upon two-photon excitation of QDs. It should be pointed out that, in the QD–PM complexes, one-photon exciting radiation is absorbed not only by the donor (QD), but also by the acceptor (PM), which makes correct studies of the FRET process difficult. In contrast, two-photon excitation is highly selective for QDs, being poorly absorbed by the PM component of the material. This allows one to investigate the fine details of the FRET mechanism in complicated nanobiological complexes and, probably, find a way to improve the photovoltaic and photochromic properties of the QD–PM hybrid system. This provides the basis for a variety of applications for this promising material. In addition, two-photon FRET is important for the development of advanced spectroscopic and microscopic imaging approaches for in vitro and in situ applications [18–20]. We have demonstrated that one- and two-photoninduced FRETs occur in the nano–bio hybrid system
Fig. 4. Dependences of (a) fluorescence intensity and (b) average fluorescence lifetimes) on the bR-to-QD molar ratio in the complexes of QDs with PMs upon excitation at 790 nm. The data obtained for the negative control (complexes of QDs with WMs) are also shown.
April 1, 2015 / Vol. 40, No. 7 / OPTICS LETTERS
formed through electrostatic assembling of QDs and PMs. We have shown experimentally that electrostatically bound QD–PM complexes are formed in this system by analysis of the dependence of the FRET efficiency on the solution concentration. We have also demonstrated selective excitation of QDs (in QD–PM complexes) in the infrared spectral region in a two-photon mode. This holds the potential to enhance the bR spectral range and sensitivity beyond the band in which the protein itself can utilize light energy. This study was supported by the Ministry of Education and Science of the Russian Federation, State Contract no. 4.624.2014/K. The part of this study dealing with the synthesis and one-photon characterization of QDs was supported by the Russian Science Foundation, grant no. 14-13-01160. References 1. N. Bouchonville, A. Le Cigne, A. Sukhanova, M. Molinari, and I. Nabiev, Laser Phys. Lett. 10, 085901 (2013). 2. A. D. Yoffe, Adv. Phys. 50, 1 (2001). 3. R. Wargnier, A. V. Baranov, V. G. Maslov, V. Stsiapura, M. Artemyev, M. Pluot, A. Sukhanova, and I. Nabiev, Nano Lett. 4, 451 (2004). 4. D. Oesterhelt, Curr. Opin. Struct. Biol. 8, 489 (1998). 5. M. D. Archer and J. Barber, Molecular to Global Photosynthesis (Imperial College, 2004). 6. A. Rakovich, A. Sukhanova, N. Bouchonville, E. Lukashev, V. Oleinikov, M. Artemyev, V. Lesnyak, N. Gaponik, M. Molinari, M. Troyon, Y. P. Rakovich, J. F. Donegan, and I. Nabiev, Nano Lett. 10, 2640 (2010).
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7. N. Bouchonville, M. Molinari, A. Sukhanova, M. Artemyev, V. A. Oleinikov, M. Troyon, and I. Nabiev, Appl. Phys. Lett. 98, 013703 (2011). 8. D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003). 9. H. Hafian, A. Sukhanova, M. Turini, P. Chames, D. Baty, M. Pluot, J. H. M. Cohen, I. Nabiev, and J.-M. Millot, Nanomedicine 10, 1701 (2014). 10. R. R. Birge, P. A. Fleitz, A. F. Lawrence, M. A. Masthay, and C. F. Zhang, Mol. Cryst. Liq. Cryst. 189, 107 (1990). 11. A. Baranov, Y. Rakovich, J. Donegan, T. Perova, R. Moore, D. Talapin, A. Rogach, Y. Masumoto, and I. Nabiev, Phys. Rev. B 68, 1653061 (2003). 12. A. Sukhanova, K. Even-Desrumeaux, A. Kisserli, T. Tabary, B. Reveil, J. M. Millot, P. Chames, D. Baty, M. Artemyev, V. Oleinikov, M. Pluot, J. H. M. Cohen, and I. Nabiev, Nanomedicine 8, 516 (2012). 13. I. E. Borissevitch, J. Lumin. 81, 219 (1999). 14. S.-C. Pu, M.-J. Yang, C.-C. Hsu, C.-W. Lai, C.-C. Hsieh, S. H. Lin, Y.-M. Cheng, and P.-T. Chou, Small 2, 1308 (2006). 15. N. S. Makarov, M. Drobizhev, and A. Rebane, Opt. Express 16, 4029 (2008). 16. J. Jasieniak, L. Smith, J. V. Embden, P. Mulvaney, and M. Califano, J. Phys. Chem. C 113, 19468 (2009). 17. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006). 18. A. Feofanov, S. Sharonov, F. Fleury, I. Kudelina, and I. Nabiev, Biophys. J. 73, 3328 (1997). 19. V. Stsiapura, A. Sukhanova, A. Baranov, M. Artemyev, O. Kulakovich, V. Oleinikov, M. Pluot, J. H. M. Cohen, and I. Nabiev, Nanotechnology 17, 581 (2006). 20. V. V. Tarabara, I. R. Nabiev, and A. V. Feofanov, Langmuir 14, 1092 (1998).
OPTICS LETTERS / Vol. 40, No. 7 / April 1, 2015
Two-photon-induced Förster resonance energy transfer in a hybrid material engineered from quantum dots and bacteriorhodopsin Victor Krivenkov,1,2,* Pavel Samokhvalov,1 Daria Solovyeva,1 Regina Bilan,1 Alexander Chistyakov,1,2 and Igor Nabiev1,3,4 1
Laboratory of Nano-Bioengineering of National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation 2 Department of Micro- and Nanophysics of National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation 3 Laboratoire de Recherche en Nanosciences, LRN-EA4682, Université de Reims Champagne-Ardenne, 51 rue Cognacq Jay, 51100 Reims, France 4 e-mail: [email protected] *Corresponding author: [email protected] Received January 16, 2015; revised February 25, 2015; accepted March 2, 2015; posted March 3, 2015 (Doc. ID 230560); published March 27, 2015 Energy transfer from nanostructures to biological supramolecular photosystems is an important fundamental issue related to the possible influence of nanoobjects on biological functions. We demonstrate here two-photon-induced Förster resonance energy transfer (FRET) from fluorescent CdSe/ZnS quantum dots (QDs) to the photosensitive protein bacteriorhodopsin (bR) in a QD–bR hybrid material. The two-photon absorption cross section of QDs has been found to be about two orders of magnitude larger than that of bR. Therefore, highly selective two-photon excitation of QDs in QD–bR complexes is possible. Moreover, the efficiency of FRET from QDs to bR is sufficient to initiate bR photoconversion through two-photon excitation of QDs in the infrared spectral region. The data demonstrate that the effective spectral range in which the bR biological function is excited can be extended beyond the band where the protein itself utilizes light energy, which could open new ways to use this promising biotechnological material. © 2015 Optical Society of America OCIS codes: (160.1435) Biomaterials; (160.4236) Nanomaterials; (160.5335) Photosensitive materials; (260.2160) Energy transfer; (020.4180) Multiphoton processes. http://dx.doi.org/10.1364/OL.40.001440
The development of novel nano-hybrid structures for light-harvesting, photovoltaic, and light-emitting devices [1] is a promising field of application of fluorescent semiconductor nanocrystals or quantum dots (QDs) [2]. Energy transfer from and between QDs plays an important role in nano–bio hybrid structures based on QDs and photosensitive biomolecules [3]. Bacteriorhodopsin (bR), a unique photosensitive protein, is known to express its photovoltaic properties upon excitation with visible light [4]. bR exposed to radiation of the solar spectrum utilizes as little as 0.1%–0.5% of the solar energy [5]. It has been shown that the use of QDs, which absorb light in a wide range, including the ultraviolet (UV) region strongly increases the efficiency of bR photovoltaic bioconversion because of single-photon Förster resonance energy transfer (FRET) from QDs to the protein [6,7]. However, single-photon excitation in QD–bR complexes is nonselective, because irradiation in the visible spectral region excites both components of the hybrid system (bR and QDs). The two-photon absorption cross section (TPACS) of QDs [8,9] is two orders of magnitude larger than maximal TPACS of bR (290 GM) [10]. This means that two-photon excitation of a QD–bR hybrid material may, first, ensure a high selectivity of QD excitation in QD–bR complexes and, second, make it possible to initiate the bR biological function in the infrared region, which would open new ways to use this material. The purpose of this study was to prepare and characterize tight self-assembled complexes of bR and QDs and to analyze the efficiencies of single-photon-excited and 0146-9592/15/071440-04$15.00/0
two-photon-excited FRETs between them in this hybrid material. The main goal of the study was to estimate the possibility of initiation of the bR biological function through two-photon-induced FRETs from fluorescent CdSe/ZnS QDs to bR in the QD–bR complexes. The core/shell CdSe/ZnS QDs for our experiments were synthesized by the hot injection method [11] and then made water soluble by replacing the surface ligand TOPO with the SH-polyethyleneglycol-OH (SH-PEG-OH) polymer as described earlier [12]. The fluorescence maxima of these QDs were at 570 nm and coincide with the absorption maxima of bR (Fig. 1). The QD QY was 14%, and the overlap integral for the QD–bR pair was 6.1 · 1015 M−1 cm−1 nm4 . We used bR in its native purple
Fig. 1. Normalized QD fluorescence and absorption spectra, and the normalized PM absorbance spectrum. © 2015 Optical Society of America
April 1, 2015 / Vol. 40, No. 7 / OPTICS LETTERS
membranes (PMs) extracted from the bacteria H. salinarum by the standard procedure [4]. White membranes (WMs) were prepared by carefully extracting the only photosensitive chromophore (retinal, the FRET acceptor) from the PMs [7]. Two-photon excitation of the samples was performed by a femtosecond laser system at a wavelength of 790 nm, pulse energy of 5 nJ, and pulse duration of 300 fs. The laser beam was focused with a 10 cm focal length lens. One-photon excitation was performed by means of the same system at a wavelength of 395 nm, pulse energy of 0.15 nJ, and pulse duration of 300 fs. To eliminate the influence of the inner filter effect on the fluorescence intensity on an increase in the acceptor concentration in our samples, the data were corrected using the correction factor calculated as described in [13]. The TPACS of QDs was calculated on the basis of comparative measurements of two-photon-induced fluorescence of nanocrystals and reference organic dyes with known TPACS as described in [14]. Fluorescein and Rhodamine 6G were used as reference dyes for TPACS determination [15]. The concentrations of QDs in solutions were estimated using the extinction coefficients in the first exciton maxima for QDs of different diameters determined in [16]. Prior to experiments on two-photon excitation of QD– PM systems, we had to test whether the nano–bio hybrid complexes of QDs and PMs were really formed and prove the possibility of one-photon FRET in our system. To do this, we monitored the FRET efficiency parameter in water mixtures of bR and QDs at different bR-to-QD molar ratios. The FRET efficiency on the experiment was calculated by comparing the fluorescence quantum yield (QY) of QDs in water with the QY of QDs mixed with a suspension of PMs. The equation for the FRET efficiency–E was obtained by substituting QY parameters for fluorescence lifetimes in Equation 13.14 from [17]: E 1 − φ∕φ0 , where φ is the QY of the QD in the presence of the PM, and φ0 is the QY of the QD in the absence of the PM. It is worth mentioning that the degradation of QY of QDs in QD–bR complexes may be related not only to the FRET from QD to bR, but also to other quenching mechanisms. To test whether FRET was the main QD quenching mechanism in our samples, we have compared the time courses of QD fluorescence quenching in QD–PM complexes and the complexes of QDs and WMs. WMs may serve as an excellent negative control for the evaluation of FRET efficiency in hybrid materials. WMs and PMs have exactly the same physical, chemical, and structural properties (lipid composition, surface charge, fluidity, rigidity, etc.). Hence, they self-assemble and form the complexes of exactly the same structure and morphology with QDs. The only difference between the QD–PM and QD–WM complexes is that FRET is impossible in the QD–WM complexes because of the absence of the FRET acceptor (retinal). To test whether the FRET occurred in QD–PM complexes upon one-photon excitation, we gradually added aliquots of a PM suspension to a water solution of QDs with a molar concentration of 2 · 10−6 M and measured the average fluorescence lifetimes (AFLs) and integrated fluorescence intensities of the resultant solutions (Fig. 2). An increase in the protein concentration in the QD–PM
1441
Fig. 2. Dependences of (a) fluorescence intensity and (b) average fluorescence lifetimes on the bR-to-QD molar ratio in the complexes of QDs with PMs upon the excitation at 395 nm. The data obtained for the negative control (complexes of QDs with WMs) are also shown.
system caused a pronounced decrease in both fluorescence intensity and AFL. In the QD–WM system, this decrease was much smaller. The difference between the samples containing PMs and WMs can be explained by assuming a zero overlap integral for the QD–WM system, in which the acceptor is absent. These data clearly confirm that the decrease in the fluorescence intensity and AFL observed in the QD–PM samples was determined by the FRET between QDs and bR. As we have speculated in our previous study [7], the self-assembly of QDs on the surface of a PM or WM may be determined by the electrostatic interaction of negatively charged QD surface groups and positively charged amino acid residues of bR located on the PM surface. To determine whether the FRET in our samples was because of the formation of tight QD–PM complexes or merely short distances from the free-floating donor particles (QDs) to the acceptor in the solution, we measured the dependence of the FRET efficiency on the concentration of QDs in the solution and compared it with the theoretical predictions for a free-particle system. The distance between the particle—r is inversely proportional to the cube root of the concentration—C. Thus, the distance changes from r 0 to r when the concentration changes from C 0 to C, by the following equation: r r 0 C 0 ∕C1∕3 :
(1)
Thus, regarding the free-floating particle system, by dilution of the sample, we would vary the concentrations and, consequently, the distance between the donor and acceptor in the aqueous mixtures of QDs and PM without changing their molar ratio. The FRET efficiency (E) can be expressed from [17]: E R60 R60 r 6 −1 ;
(2)
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OPTICS LETTERS / Vol. 40, No. 7 / April 1, 2015
where R0 is the Forster radius. Using Eqs. (1) and (2), we can estimate the dependence of the E on the C by the following equation: E R60 R60 r 60 C 0 ∕C2 −1 ;
(3)
where C 0 is the solution concentration when the distance between particles is r 0 and the FRET efficiency is known and equal to E 0 . R0 does not change with changing concentration; hence, it can be expressed in terms of r 0 and E0 using Eq. (2): R0 r 0 E0 1 − E 0 −1 1∕6 :
(4)
Thus, by substituting R0 from Eq. (4) into Eq. (3), we obtain the final expression for the dependence of the FRET efficiency on the concentration of the solution: 2 −1 E 1 E −1 0 1 − E 0 C 0 ∕C :
(5)
Next, we measured the experimental dependence of the FRET efficiency on the concentration of solution, to compare with the theoretical dependence and validate our assumption that the system is not the set of free-floating particles, but it is an aqueous solution of the QD-bR complexes. To obtain the experimental dependence of the FRET efficiency from the solution concentration, we used an aqueous mixture of QDs and PMs with an initial FRET efficiency (E 0 ) of 0.4, a bR-to-QD molar ratio of 4, and a QD concentration of 2 · 10−7 M. The stock solution was diluted sequentially 16 times, and the QY was measured at each dilution step. Figure 3 shows the theoretical and experimental dependences of the FRET efficiency on the concentration of the solution. It is clear that the FRET efficiency is much higher than could be expected from the model of free-floating particles at every experimental point. The observed decrease in the FRET efficiency (from 0.4 to 0.25 after 16-fold dilution of the sample) can be explained by assuming that the complexes were formed upon adsorption of QDs onto PMs, and there was a dynamic equilibrium between the desorbed QDs and QD–PM complexes in the solutions studied. Although efficient one-photon-induced FRET in QD– bR hybrid materials has been demonstrated earlier [1,6,7], two-photon-induced FRET from a nanomaterial to a photosensitive biological system has never been observed before to the best of our knowledge. The possibility to induce or enhance a biological function through excitation in an optical region wider than the spectral
Fig. 3. Experimental FRET efficiency upon dilution of the QD–bR sample and the theoretically predicted FRET efficiency for a free-floating particle system Eq. (5).
range where natural biosystems utilize light energy may open new prospects for biophotonic applications and the development of bioinspired devices. Therefore, we have further analyzed the possibility of efficient two-photoninduced FRETs in tight QD–PM complexes developed, and structurally and optically characterized above. First, we calculated the TPACS of our QDs and found it to be two orders of magnitude higher than that of bR. The measured value of the QD TPACS at an excitation wavelength of 790 nm was about 20,000 GM. The FRET in the QD–PM system upon two-photon excitation was induced in a manner similar to that of one-photonexcitation described above. The dependences of the fluorescence intensity and AFL on the bR-to-QD molar ratio are shown in Fig. 4. It can be seen that an increase in the PM concentration leads to a decrease in the fluorescence intensity and AFL in the QD–PM system. As in the case of one-photon excitation, this decrease was much smaller in the QD–WM system. These data show that FRET occurs in QD–PM hybrid complexes upon two-photon excitation of QDs. It should be pointed out that, in the QD–PM complexes, one-photon exciting radiation is absorbed not only by the donor (QD), but also by the acceptor (PM), which makes correct studies of the FRET process difficult. In contrast, two-photon excitation is highly selective for QDs, being poorly absorbed by the PM component of the material. This allows one to investigate the fine details of the FRET mechanism in complicated nanobiological complexes and, probably, find a way to improve the photovoltaic and photochromic properties of the QD–PM hybrid system. This provides the basis for a variety of applications for this promising material. In addition, two-photon FRET is important for the development of advanced spectroscopic and microscopic imaging approaches for in vitro and in situ applications [18–20]. We have demonstrated that one- and two-photoninduced FRETs occur in the nano–bio hybrid system
Fig. 4. Dependences of (a) fluorescence intensity and (b) average fluorescence lifetimes) on the bR-to-QD molar ratio in the complexes of QDs with PMs upon excitation at 790 nm. The data obtained for the negative control (complexes of QDs with WMs) are also shown.
April 1, 2015 / Vol. 40, No. 7 / OPTICS LETTERS
formed through electrostatic assembling of QDs and PMs. We have shown experimentally that electrostatically bound QD–PM complexes are formed in this system by analysis of the dependence of the FRET efficiency on the solution concentration. We have also demonstrated selective excitation of QDs (in QD–PM complexes) in the infrared spectral region in a two-photon mode. This holds the potential to enhance the bR spectral range and sensitivity beyond the band in which the protein itself can utilize light energy. This study was supported by the Ministry of Education and Science of the Russian Federation, State Contract no. 4.624.2014/K. The part of this study dealing with the synthesis and one-photon characterization of QDs was supported by the Russian Science Foundation, grant no. 14-13-01160. References 1. N. Bouchonville, A. Le Cigne, A. Sukhanova, M. Molinari, and I. Nabiev, Laser Phys. Lett. 10, 085901 (2013). 2. A. D. Yoffe, Adv. Phys. 50, 1 (2001). 3. R. Wargnier, A. V. Baranov, V. G. Maslov, V. Stsiapura, M. Artemyev, M. Pluot, A. Sukhanova, and I. Nabiev, Nano Lett. 4, 451 (2004). 4. D. Oesterhelt, Curr. Opin. Struct. Biol. 8, 489 (1998). 5. M. D. Archer and J. Barber, Molecular to Global Photosynthesis (Imperial College, 2004). 6. A. Rakovich, A. Sukhanova, N. Bouchonville, E. Lukashev, V. Oleinikov, M. Artemyev, V. Lesnyak, N. Gaponik, M. Molinari, M. Troyon, Y. P. Rakovich, J. F. Donegan, and I. Nabiev, Nano Lett. 10, 2640 (2010).
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