© Copyright 2006 American Chemical Society
OCTOBER 10, 2006 VOLUME 22, NUMBER 21
Letters Modulating Fluorescence Resonance Energy Transfer in Conjugated Liposomes Xuelian Li, Matthew McCarroll, and Punit Kohli* Department of Chemistry and Biochemistry, Southern Illinois UniVersity, Carbondale, Illinois 62901 ReceiVed May 11, 2006. In Final Form: August 17, 2006 We report here a novel system where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism.
Fluorescence resonance energy transfer (FRET) is a process whereby energy is nonradiatively transferred from a donor molecule to an acceptor molecule through long-range dipole interactions. Recently, FRET has been exploited for many applications including DNA sensing,1a-c proteomics,1d and probing various processes in living cells.1e,f These applications all rely on the remarkable dependence of the FRET efficiency on the donor-acceptor separation distance. Most FRET-based sensing schemes are based on the modulation of the separation distance to trigger a sensing response. The use of the other factors in the Fo¨rster equation, such as spectral overlap and donoracceptor dipole orientation, have been underutilized in the development of sensing systems.2 The goal of the work reported here is to investigate FRET (the feasibility of building sensing systems) based primarily on the modulation of the spectral overlap and acceptor quantum yield. * Corresponding author. E-mail:
[email protected]. (1) (a) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (b) Zhang, P.; Beck, T.; Tan, W. Angew. Chem., Int. Ed. 2001, 40, 402-405. (c) Liu, B.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 589-593. (d) Kumaraswamy, S.; Bergstedt, T.; Shi, X.; Rininsland, F.; Kushon, S.; Xia, W.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511-7515. (e) Zal, T.; Gascoigne, N. R. J. Biophys. J. 2004, 86, 3923-39. (f) Verveer, P. J.; Wouters, F. S.; Reynolds, A. R.; Bastiaens, P. I. H. Science 2000, 290, 1567-1570. (2) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B. R.; Bawendi, M. G.; Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301-310.
The rate of energy transfer, kET, between a donor and an acceptor molecule is given by
kET )
Kk2QJ r6
(1)
where K is a constant, k2 is an orientation factor between donor and acceptor molecules, Q is the quantum yield of the donor, J is the overlap between the donor emission spectrum and absorption acceptor spectrum, and r is the donor-acceptor distance.3 The wide success of FRET-based sensing systems is based on the r6 dependence of kET that results in extremely large differences between the “on” and “off” states of the sensor. Although the distance dependence is the dominant term in eq 1, kET is also linearly dependent on J, Q, and k2. We report here a novel system that utilizes changes in J and changes in the quantum yield of the acceptors for fluorescence amplification for the rate of energy transfer between donor and acceptor. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET through conformationally induced changes in the PDA absorption spectrum. Polydiacetylenes are intriguing conjugated polymers because their electronic absorption spectrum changes dramatically in (3) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/ Plenum: New York, 1999; Chapter 13.
10.1021/la061340m CCC: $33.50 © 2006 American Chemical Society Published on Web 09/13/2006
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Figure 1. (A) Structures of two monomers used in this study. (B) Corrected normalized absorption absorption spectra of blue- (blue curve) and red-form (red curve) PDA liposome solutions (1 mM) at 298 K. The green curve is the emission spectrum of the PDA liposome solution composed of monomers 1 and 2 (mol ratio [2]/[1] ) 1000, [monomertotal] ) 1 mM).
response to stress applied to the conjugated ene-yne backbone and can exist in either a blue or red form. These polymeric systems have been used as liposome-based colorimetric sensors for the detection of influenza viruses,4a cholera toxins,4b antimicrobial peptides,4c and antibody-antigen interactions.4d In this work, we have chosen to use PDA as the FRET acceptor because the donor-acceptor overlap integral will be modulated in response to changes in the absorption spectrum. As a proof of concept, the conversion from the blue form to the red form can be accomplished by heating.5 We have chosen dansyl and PDA as the donor-acceptor pair for the following reasons: (1) The spectral overlap between dansyl emission and PDA absorption in its blue form is relatively small and increases significantly in the red form when PDA is transformed into its red form (Figure 1B). (2) Dansyl emission is sensitive to the local environment.3 This solvent-sensitive emission property of dansyl fluorophores can be utilized to probe the local environment of bilayer PDA liposomes during heating of the PDA liposomes. (3) The background emission from the blue form of PDA is very low because of an extremely small quantum yield (∼10-4). Importantly, in the red form, the quantum yield of PDA increases nearly 2 orders of magnitude to a value of 0.02.6 These changes in the quantum yield of PDA increase the value of this emission shift in sensing applications. We have produced a FRET platform with a relatively fixed donor-acceptor distance by synthesizing bilayered liposomes following the self-assembly of a mixture of dansyl-tagged 10,12pentacosdiyoic acid (1) and 10,12-pentacosdiyoic acid (2) monomers (Figure 1A).8 Dialysis results indicate that >95% of all dansyl-tagged fluorophores were covalently bound in the liposomes.8 Figure 2A shows the absorption spectrum of PDA at various temperatures. The dominant peak centered at 630 nm results from the blue-form PDA at 298 K.4,5 As the temperature of the PDA solution is increased, the intensity of the peak at 630 nm decreased while two distinct peaks (centered at 495 and 540 nm) (4) (a) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585-588. (b) Pan, J.; Charych, D. Langmuir 1997, 13, 1365-1367. (c) Kolusheva, S.; Boyer, L.; Jelinek, R. Nat. Biotechnol. 2000, 18, 225-227. (d) Kolusheva, S.; Kafri, R.; Marina, K.; Jelinek, R. J. Am. Chem. Soc. 2001, 123, 417-422. (5) (a) Okada, S.; Peng, S.; Spevak, W.; Charych, D. Acc. Chem. Res. 1998, 31, 229-239. (b) Hofmann, U. G.; Peltonen, J. Langmuir 2001, 17, 1518-1524. (c) Carpick, R. W.; Mayer, T. M.; Sasaki, D. Y.; Burns, A. R. Langmuir 2000, 16, 1270-1278. (6) Olmsted, J., III.; Strand, M. J. Phys. Chem. 1983, 87, 4790-4792.
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Figure 2. (A) UV-vis and (B) fluorescence responses (λex ) 337 nm) of a liposome solution composed of ([2]/[1] ) 1000, [monomertotal] ) 1 mM) for different thermal treatments. The insets in A are photographs of vials containing blue- and red-form PDA. UVvis and fluorescence spectra were taken at 298 K by first heating the liposome solutions to a given temperature for 10 min followed by equilibrating the solution to 298 K for 10 min. The dotted curve in B is the emission spectrum of the same solution but without dansyl fluorophores.
whose intensities grow with increasing temperature of the solution were observed. In fact, we observe an isosbestic point at a wavelength of 555 nm (Figure 2A) that strongly suggests that there are two species and that thermal-induced stress leads to the conversion of the PDA blue form to the PDA red form. These observations are consistent with previous reports.5 The overlap between the absorption spectrum of PDA and the emission spectrum of dansyl (J) increases with thermal treatment, ultimately leading to an increase in the FRET efficiency (kET) from dansyl to PDA (eq 1). For example, we have experimentally determined that the overlap intergral (J) increases from a value of 1.1 × 1010 M-1 cm-1 nm4 in the blue form to 2.52 × 1010 M-1 cm-1 nm4 in the red form, following thermal treatment. This represents an ∼130% increase in the J value.7 Dansyl is a solvent-polarity-sensitive probe whose emission wavelength can provide useful information regarding its local environment in the liposome bilayers.3 The emission spectrum for the dansyl-containing liposomes is an anomalously broad peak centered at 484 nm, indicating that the dansyl fluorophores are located in the liposome solution with environments ranging from nonpolar to those of moderate polarities (Figure 2B). Following conversion to the red form, the broad emission spectrum becomes more well defined with peaks appearing at 465, 519, and 560 nm (Figure 2B). The peaks at 465 and 519 nm can be attributed to the dansyl moiety existing in one of two welldefined environments of differing polarities, corresponding to those of hexane and ethanol, respectively. The peak at 560 nm corresponds to the emission of the red-form PDA and can be tentatively attributed to the FRET process. The magnitude of the increased intensity of the peak at 560 nm is dependent on the temperature of the thermal treatment. In fact, there was a modest (7) The overlap function, J(λ), between dansyl fluorophore emission and PDA absorption (Figure 1) is calculated using the following equation (see chapter 13 of ref 3 for more details): J(λ) ) ∫ F(λ) (λ)λ4δλ/∫ F(λ)δλ, where F(λ) is the fluorescence intensity of the donor (dimensionless), (λ) is the extinction coefficient (M-1 cm-1), and λ is the wavelength (nm). J(λ) is in M-1 cm-1 nm.4 (8) Supporting information accompanying this communication.
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increase (∼2×) when the solution was treated at 328 K, whereas the intensity increased 10-fold after treating the liposome solution at 343 K (Figure 2B). The peak centered at ∼560 nm is attributed to the PDA emission5b,c,9 following the FRET from dansyl donors to PDA acceptors (vide infra). To support this conclusion, we performed two control experiments. First, the direct excitation at 470 nm of liposomes shows an emission peak centered at ∼560 nm that increases in intensity following incremental thermal treatment of the solution (Figure 1S, Supporting Information). The liposomes were derived from monomers of [1] and [2] (mol ratio [2]/[1] ) 1000, [monomertotal] ) 1 mM). There is reasonable assurance that dansyl does not contribute to the observed fluorescence because of its extremely low molar extinction coefficient at 470 nm10 and the fact that the 560 nm peak (Figure 2B) is indeed from FRET-derived PDA fluorescence. Second, when the same liposome solution without dansyl is excited in a manner similar to that used in the FRET experiment (λex ) 337 nm), the direct PDA emission was ∼20 times smaller than that of the similar solution containing both donor and acceptor (Figure 2B). These controlled experiments strongly support the conclusion that the emission peak at 560 nm is from PDA emission as a result of the FRET process. Interestingly, in our FRET experiments the changes in the emission intensity of the donor and acceptor were not proportional, as is the case in typical FRET. Following temperature treatments at increasing temperatures, the donor emission decreased to a constant value at 328 K, whereas the acceptor emission continued to increase up to 343 K (Figure 2B), indicating that an additional process affected the acceptor emission. With all other parameters being constant, the increase in J cannot alone account for the >10 times increase in the acceptor intensity. It was previously reported that the temperature treatment of a PDA solution at T > 320 K results in a considerable increase in the fraction of red-form PDA at the expense of the blue form9 and that the quantum yield of PDA is more than 2 orders of magnitude greater in the red form.6 Our observations are consistent with the literature, and we believe the large increase in the quantum yield of the red PDA partially contributes to the dramatic increase in the 560 nm emission peak. It should be noted that we have not specifically examined the effect of the donor-acceptor distance, r, and the donor quantum yield dependence on FRET in this system. Our preliminarily TEM data suggests that diacetylene liposomes are mechanical robust up to 343 K, hence we can expect the separation distance to remain relatively constant in a given liposome system. To investigate this possible effect, we are now designing (9) (a) Carpick, R. W.; Mayer, T. M.; Sasaki, D. Y.; Burns, A. R. Langmuir 2000, 16, 4639-4647. (10) The extinction coefficient of dansyl-amide in water at 470 nm is ∼18 M-1 cm-1 (Chen, R. F. Anal. Biochem. 1968, 25, 412-416).
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fluorophore-tagged diacetylene monomers where we can systematically change the donor-acceptor separation distance. We now discuss an issue of FRET versus direct PDA excitation for sensing applications. The emission of a fluorophore depends on the extinction coefficient, quantum yield of fluorophore, and instrumental parameters such as the light source, detector, and optics as well. Considering the extremely low quantum yield (∼0.02) and moderate extinction coefficient of the red-PDA form (∼4000 M-1 cm-1, depending upon polymerization conditions such as polymerization time, presence of oxygen in the solution, etc.), the emission intensity from PDA through direct PDA excitation would be extremely low, especially for concentration
