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Langmuir 2003, 19, 3554-3556
Photoluminescence Quenching of Water-Soluble Conjugated Polymers by Viologen Derivatives: Effect of Hydrophobicity
Chart 1
Chunhai Fan,† Takashi Hirasa,†,‡ Kevin W. Plaxco,†,§ and Alan J. Heeger*,†,⊥ Institute for Polymers and Organic Solids, Department of Chemistry and Biochemistry, and Interdepartmental Biomolecular Science and Engineering Program, and Physics Department and Materials Department, University of California at Santa Barbara, Santa Barbara, California 93106, and Science & Technology Research Center, Mitsubishi Chemicals Corporation, Japan Received October 3, 2002. In Final Form: January 27, 2003
Introduction Conjugated (semiconducting) polymers1 have potential utility in optical and electronical sensors for peptides, proteins, and nucleic acids.2-8 Chen et al. recently developed a fluorescent biosensor involving a watersoluble conjugated poly[lithium 5-methoxy-2-(4-sulfobutoxy)-1,4-phenylenevinylene] (MBL-PPV, Chart 1).9 They demonstrated that the luminescence of MBL-PPV is quenched by the electron acceptor methyl viologen (MV2+) with extraordinary efficiency (“superquenching”). Because of this superquenching, even minute amounts of quencher induce easily detectable decreases in polymer luminescence. To create a novel, highly sensitive class of biosensors, MV2+ was tethered to a ligand such that the quencher is sequestered upon binding to a specific, biologically related target. In the first example explored, biotin was employed as the ligand in a quencher-tether-ligand (QTL) construct such that luminescence is restored (“turned on”) when traces of avidin, the binding partner of biotin, are present. In principle, QTL provides a versatile platform for a wide range of biorecognition-based sensors. The sensitivity of PPV-based biosensors is directly related to the superquenching efficiency of the MBL-PPV/ MV2+ pair. Quenching efficiency can be quantified through measurements of the Stern-Volmer constant, Ksv:
φ°/φ ) 1 + Ksv[quencher]
(1)
where φ° and φ are the photoluminescence (PL) quantum * Corresponding author. E-mail:
[email protected]. † Institute for Polymers and Organic Solids. ‡ Mitsubishi Chemicals Corp. § Department of Chemistry and Biochemistry and Interdepartmental Biomolecular Science and Engineering Program. ⊥ Physics Department and Materials Department. (1) Heeger, A. J. Nobel Lecture, 2000; http://www.nobel.se. (2) Fan, C.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124, 5642. (3) Heeger, P. S.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12219. (4) Gerard, M.; Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2001, 17, 345. (5) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548. (6) Kumpumbu-Kalemba, L.; Leclerc, M. Chem. Commun. 2000, 1847. (7) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954. (8) Leclerc, M. Adv. Mater. 1999, 1491. (9) Chen, L.; McBranch, D. W.; Wang, H.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287.
efficiencies in the absence and presence of the quencher, respectively. Thus, in general, the higher the Ksv, the lower the concentration of quencher required to achieve a given level of PL quenching and the greater the detection sensitivity. As a result of our attempts to identify systems with still higher Ksv, we recently demonstrated that the electron transfer protein, cytochrome c, acts as an excellent quencher with significantly increased Ksv. More importantly, with cytochrome c, Ksv can be varied over a wide range by changing the protein’s charge state (by changing, for example, the pH).2 The extremely high Ksv of the MBL-PPV/MV2+ pair is thought to derive from a combination of several effects. The delocalized electronic structure of conjugated polymers enables efficient light harvesting and ultrafast photoinduced electron transfer. In addition, the oppositely charged polymer and quencher form a weak electrostatic complex. As a result, static quenching via ultrafast photoinduced electron transfer dominates the quenching mechanism.10 Aggregation of MBL-PPV upon complex formation in water also contributes to the high quenching efficiency.11 Additional studies of the MBL-PPV/viologen pair are of interest with the goal of obtaining higher Ksv though derivatization of viologen. Studies at the University of California at Santa Barbara (UCSB) showed that the quenching efficiency of viologen derivatives is strongly dependent on the net charge.12 This effect has been substantiated by research on other charged polymer/ quencher systems.11,13-15 In contrast, the effect of hydrophobicity on the quenching efficiency of viologen derivatives has been largely neglected. Here we characterize the effect of changing quencher hydrophobicity on the quenching of polymer luminescence. Materials and Methods To probe the effect of quencher hydrophobicity on quenching efficiency, we have employed a series of viologen derivatives of increasing side chain length. The four viologens, methyl viologen (MV2+), ethyl viologen (EV2+), hexyl viologen (HV2+), and benzyl viologen (BV2+) (Chart 2), contain an identical bipyridinium group. MV2+, EV2+, and BV2+ were obtained from Sigma without further purification. HV2+ was synthesized by mixing 4-4′dipyridine and excess 1-iodohexane in dimethylformamide (10) Wang, J.; Wang, D.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J. Macromolecules 2000, 33, 5153. (11) Tan, C.; Pinto, M. R.; Schanze, K. S. Chem. Commun. 2002, 446. (12) Wang, D.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J. Langmuir 2001, 17, 1262. (13) Lu, L.; Helgeson, R.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 124, 483. (14) Place, I.; Perlstein, J.; Penner, T. L.; Whitten, D. G. Langmuir 2000, 16, 9042. (15) Harrison, B. S.; Ramey, M. B.; Reynolds, J. R.; Schanze, K. S. J. Am. Chem. Soc. 2000, 122, 8561.
10.1021/la026651l CCC: $25.00 © 2003 American Chemical Society Published on Web 03/19/2003
Notes
Langmuir, Vol. 19, No. 8, 2003 3555 Chart 2
Table 1. Fluorescence Quenching of MBL-PPV by Viologen Derivatives drop of absorption chain of E0′ (V vs absorption (at 432 nm) viologen NHE)a peak (nm) (%)
(DMF). The mixture was refluxed for 2 h and then cooled to room temperature. Filtration and extraction with dry toluene by using a Soxhlet extractor gave a orange-red solid. 1H NMR (200 MHz, D2O): 9.11 (d, 4H), 8.94 (d, 4H), 4.71 (t, 4H), 2.06 (m, 4H), 1.32 (m, 12H), 0.84 (t, 6H). Absorption spectra were collected with a UV-2401PC spectrophotometer (Shimadzu). Fluorescence spectra were recorded using a 1.0 cm path length quartz cuvette in a PTI fluorometer (Photon Technology International). The absorption spectroscopy was performed at much higher quencher concentration than that employed in the Stern-Volmer experiments due to the extremely insensitive nature of absorbance spectroscopy relative to fluorescence spectroscopy. Cyclic voltammetry was performed on a CHI 603 workstation (CH Instruments) combined with a BAS C-3 stand in an anaerobic condition. A glassy carbon (GC) working electrode, a Ag/AgCl/3 M NaCl reference electrode (BAS), and a platinum wire (BAS) were used as a normal three-electrode configuration. Potentials are reported against the normal hydrogen electrode (NHE). The GC electrode was polished to mirror smoothness with 0.03 µm alumina slurry on a polishing microcloth and then rinsed thoroughly with Nanopure water. The test solution was purged with argon and a stream of argon was maintained above it in order to keep the solution anaerobic throughout the experiment.
Results and Discussion Previous reports have confirmed that ionic quenchers enhance the aggregation of water-soluble conjugated polymers.12 We monitored the UV-vis absorption spectra of MBL-PPV, which provides direct evidence of polymer aggregation, in the absence and presence of the viologen derivatives.12 For MBL-PPV, spectral shifts and decrease
MBL-PPV MV2+ EV2+ HV2+ BV2+
C1 C2 C6 benzyl
-0.435 -0.443 -0.366 -0.321
432 444 444 442 444
3.8 6.3 8.8 10.1
Ksvb (×108) 0.59 (0.02) 1.02 (0.09) 1.92 (0.24) 2.34 (0.14)
a Data were collected at 1 mM viologen in a pH 7.4, 10 mM phosphate buffer containing 100 mM NaCl. b Reported are the mean values from three independent measurements, with standard deviations in parentheses.
of absorption peaks were observed in the presence of all of the viologen derivatives, irrespective of side chain length (Figure 1). All four viologen derivatives shift the absorption peak (10-12 nm) to the red (Table 1), consistent with the previous studies on charged viologen derivatives.12 Although the red-shift of the peak is nearly the same for the four compounds, the absorption intensity decreases in a stepwise manner as the number of carbons in the side chain increases for MV2+, EV2+, HV2+, and BV2+. Previous studies have shown that highly charged viologen derivatives such as MV4+ lead to a significant drop in UV-vis absorption which is attributed to polymer aggregation and consequent precipitation.12 For the viologen derivatives described here, however, there is no evidence of the significant changes in UV-vis absorption associated with precipitation (Table 1). Thus the changes in quenching efficiency induced by the increasingly large side chains of the viologen derivatives are more subtle than the effects arising from increasing quencher charge. Stern-Volmer studies with the viologen derivatives as quenchers quantitatively indicate that their quenching efficiencies increase as the size of the side chain increases (Figure 2 and Table 1). While all four viologen derivatives efficiently quench the MBL-PPV fluorescence, EV2+
Figure 1. UV-vis spectra of MBL-PPV in the absence and presence of the four viologen derivatives employed here show no indication of quencher-induced precipitation. The experiments are conducted at an MBL-PPV concentration of 1.5 × 10-5 M and an MBL-PPV to viologen ratio of 5.
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Figure 2. Stern-Volmer plots of MBL-PPV (1 µM) quenched by viologen derivatives: MV2+ (circle), EV2+ (triangle), HV2+ (diamond), BV2+ (square).
quenches with significantly higher Ksv than that of MV2+. The addition of a single methylene group to the side chain almost doubles quenching efficiency. Quenching efficiency is further enhanced by extending the length of the side chain; the Ksv of hexyl viologen is again twice that of ethyl viologen. Several effects might account for such an enhancement in quenching efficiency. First, elongation of the side chain increases the possibility of collision between MBL-PPV and viologen. Second, the reorganization energy for reduction of the viologen derivatives might vary with chain length. Third, the hydrophobicity of the viologen derivatives increases with chain length. The relative Ksv values of hexyl and benzyl viologen, however, suggest that differing collision rates contribute little if any to the observed changes in quenching efficiency. The benzyl group is more compact than the hexyl group, and yet the Ksv of BV2+ is slightly greater than that of hexyl viologen. Similarly, measurements of the variation of reduction potential for the viologen derivatives as a function of side chain length indicate that changes in viologen reorganization energy do not contribute significantly to their differing Ksv values. While the reduction potential (E0′) differs slightly between the various viologen derivatives, the absolute difference in E0′ is relatively small (∼0.12 V) and poorly correlated with the observed changes in Ksv (Table 1). In contrast to their reduction potentials, side chain size, and thus hydrophobicity, are highly correlated with relative quenching efficiency (Figure 3; r2 ) 0.99).
Notes
Figure 3. Ksv values of viologen derivatives are strongly correlated with the number of carbon atoms of their side chains (and thus with the hydrophobicity).
Thus it appears that differences in quencher hydrophobicity give rise to the differing quenching efficiencies of the viologen derivatives described here. Small changes in the UV-vis absorption of MBL-PPV upon the addition of the viologen derivatives provide further evidence for the role of hydrophobic interactions in generating the enhanced quenching efficiency. The observed changes in Ksv are consistent with small, stepwise decreases in the polymer’s UV-vis absorption (Table 1), suggesting that the increasing hydrophobicity of the quenchers enhances polymer aggregation, which in turn enhances quenching efficiency. Conclusion The superquenching of conjugated polymers by viologen derivatives involves numerous factors. In addition to the previously proposed effects of efficient charge transfer and electrostatic interactions, it appears that the hydrophobicity of the quencher also plays a measurable role. We expect that further studies will bring about better understanding of this complex interaction, which should in turn lead to the design of more sensitive biosensors. Acknowledgment. This research was supported by the National Science Foundation under DMR-0099843 and by the Office of Naval Research (Grant ONR N00141-1-0239) and the National Institutes of Health (Grant GM 62958-01). LA026651L
