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Superquenching and Its Applications in J-Aggregated Cyanine Polymers Robert M. Jones, Troy S. Bergstedt, C. Thomas Buscher, Duncan McBranch, and David Whitten* QTL Biosystems, LLC, Santa Fe, New Mexico 87501 Received December 12, 2000. In Final Form: February 27, 2001 We report the observation of a “superquenching” for water-soluble poly-L-lysine derivatives containing an appended cationic cyanine dye on each repeat unit. The formally nonconjugated cyanine dye chromophores strongly associate in a “J” aggregate structure characterized by a sharp red-shifted absorption (compared to the monomer) and a similarly sharp red-shifted fluorescence. Superquenching is manifested by very large “Stern-Volmer” constants for fluorescence quenching by oppositely charged electron acceptors or energy transfer dyes; substantial quenching is observed at levels of quencher corresponding to one to four molecules per polymer chain. The quenching observed for these polymers is equivalent or greater to that previously observed for conjugated polyelectrolytes. We have been able to exploit the superquenching of the “J” aggregate polymer fluorescence in a competitive bioassay.
Introduction The contrasts in reactivity between individual molecules and supramolecular assemblies continue to be the subject of much active investigation. Recently we reported on the photophysics and fluorescence quenching of a conjugated polyelectrolyte in aqueous solution; a remarkable finding was that the fluorescence of the polymer is quenched by extremely low amounts of neutral or oppositely charged electron acceptors.1-3 The high sensitivity to quenching (quantitatively measured by the Stern-Volmer quenching constant, Ksv) can be used as the basis for sensitive but not particularly selective chemical sensing. The static quenching observed is characterized by Ksv values 106fold higher than those for corresponding small molecule quenching. For quenching of the polymer by oppositely charged electron acceptors, the origin of the “superquenching” can be attributed to the combination of at least two factors. The polyelectrolyte can bind hydrophobic counterions and neutral molecules in a nonselective manner in much the same way that a micelle “collects” the same species. Additionally, the observation of strong quenching under conditions where the quencher is present at levels of 1-10 molecules per polymer chain indicates that a single quencher is effective at quenching excitation delivered to many sites within the polymer. Energy migration in the polymer can be attributable to exciton delocalization or Fo¨rster energy transfer or a combination thereof.2,4-9 We found that it was possible to use the quenching sensitivity as the basis for highly selective and sensitive biosensors by constructing conjugates containing a charged quencher linked by a short tether to a ligand (1) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. 1999, 96, 12287-12292. (2) Chen, L.; McBranch, D.; Wang, R. Whitten, D. Chem. Phys. Lett. 2000, 330, 27-33. (3) Chen, L.; Xu, S.; McBranch, D.; Whitten, D. J. Am. Chem. Soc. 2000, 122, 9302-9303. (4) Nguyen, T.-Q.; Wu, J.; Doan, V.; Schwartz, B. J.; Tolbert, S. H. Science 2000, 288, 652-656. (5) Swager, T. M. Acc. Chem. Res. 1998, 31, 201-207. (6) Rothberg, L. J.; Yan, M.; Papadimitrakopoulos, F.; Galvin, M. E.; Kwock, E. K.; Miller, T. M. Synth. Met. 1996, 80, 41. (7) Rice, M. J.; Garstein, Y. N. Phys. Rev. Lett. 1994, 73, 2504. (8) Mukamel, S.; Tretiak, S.; Wagersreiter, T.; Chernyak, V. Science 1997, 277, 781. (9) Bredas, J.-L.; Cornil, J.; Beljonne, D.; Dos Santos, D. A.; Shuai, Z. Acc. Chem. Res. 1999, 32, 267-276.
selective for a specific biomacromolecule. While the previous observations have been made with conjugated polymers having at least in principle an extended conjugation through the polymer backbone, we felt it might be possible that similar phenomena might occur with polymers containing chromophores pendant on the polymer backbone, but not in direct conjugation with one another. Previous studies have shown that such polymers often exhibit strong aggregation of the chromophores due to proximity and special spatial arrangements.10-12 In the present paper we report that a polymer containing nonconjugated cyanine chromophores appended to each repeat unit and exhibiting J-aggregation shows a “superquenching” sensitivity equal to or greater to that observed with the conjugated polymers previously studied. Remarkably this polymer exhibits high quenching sensitivity both in a variety of solution environments and when transferred to certain charged supports. Experimental Section Quenching Experiments. The polymer used in this study is synthesized by building a cationic cyanine dye onto a polyL-lysine “scaffold”. The polymer (1) has been synthesized and purified as reported elsewhere.10,11 The poly-L-lysine (hydrobromide) used in this study has a molecular weight in the range 50-60 kD and thus contains ∼250 repeat units per chain. Aqueous solutions of the cationic dye-polymer 1 used for emission measurements were prepared by diluting (∼5000-fold) DMF stock solutions of the polymer to yield optical densities of less than 50 mOD in a 1 cm path length quartz cuvette. Laponite RDS clay was obtained from Southern Clay Products and prepared as aqueous suspensions at concentrations ranging between 10-3 and 1% (w/v). Preparations of 1 suspensions on Laponite clay were obtained by titrating solutions of 1 with clay suspensions. Progress of the deposition of 1 on clay was followed spectrophotometrically to ensure complete uptake of the polymer while avoiding the presence of excess uncoated clay particles. All quenchers were prepared as aqueous solutions at 1001000 times the molar concentration of the solutions of 1. At these high concentrations addition of quencher to polymer solutions resulted in negligible dilution of the sample. All fluorescence (10) Place, I.; Perlstein, J.; Penner, T. L.; Whitten, D. G. Langmuir 2000, 16, 9042-9048. (11) Roberts, M. R.; Coltrain, B. C.; Melpolder, S. M.; Wake, R. W. Ceram. Trans. 1991, 19, 287. (12) Fox, M. A. Acc. Chem. Res. 1999, 32, 201-207.
10.1021/la001745l CCC: $20.00 © 2001 American Chemical Society Published on Web 04/04/2001
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Figure 1. Absorption and emission spectra of dye polymers in water: solid trace, polymer 4 absorption; dashed trace, polymer 4 fluorescence; dotted trace, polymer 1 absorption; dot-dash trace, polymer 1 fluorescence. Table 1. Quenching of Polymer Fluorescence by Oppositely Charged Ions polymer
quencher
medium
Ksv (M-1)
PRU/Qa at 50% quenched
1 1 1 1 4 4 4
2 2 2 3 MV2+ c MV2+ MV2+
H2O PBSb-H2O 50/50 H2O-DMSO H2O H 2O PBSb-H2O 50/50 H2O-DMSO
7 × 107 2 × 107 8 × 107 4 × 107 2 × 107 7.7 × 105 4.7 × 105
133 14.4 154 60 20 0.18 0.76
c
a Polymer repeat unit per quencher. b Phosphate-buffered saline. Methyl viologen N,N′-dimethyl-4,4′-bipyridinium.
measurements were acquired at 20 ( 0.1 °C using a Spex Fluorolog spectroluorimeter. Biosensing Experiment. A competitive assay involving the avidin-biotin receptor-ligand sytem was conducted as follows. A 500-µL portion of 0.1 µM 1 was added to the filtrate (lower) chamber of each of three separate Microcon YM-30 centrifugal filter vials. In each of these devices the filtrate chamber is separated from the retentate (upper) chamber by a 30 kDa nominal molecular weight cutoff (NMWCO) cellulose membrane. This membrane is impermeable to the 66 kDa protein avidin. The three retentate chambers were charged with (a) 0.5 µM D-biotin and (b and c) 0.8 µM AQS-biotin conjugate + 1.0 µM avidin, respectively. The three tubes were then each sealed and incubated for 10 min, after which time 0.5 µM D-biotin was added to vial c. All three vials were then centrifuged for 1 min at 12000g to allow permeable species in the retentates to mix with the filtrate containing 1. The fluorescence emission of all three filtrates was then measured at 20.0 ( 0.1 °C on a SPEX FluoroMax-3.
Results and Discussion In both aqueous solution and on certain solid supports polymer 1 exhibits extremely sharp absorption and fluorescence spectra (with a very small Stokes shift) characteristic of “J” aggregation (Figure 1).10 The absorption is shifted to the red 67 nm compared to the monomer dye. Since the cyanine polymer has a net positive charge, the anionic anthraquinone disulfonate (2) and the negatively charged cyanine (3)13 were chosen as potential quenchers that might be effective at very low concentrations (Scheme 1). When dilute (∼2 × 10-6 M in repeat unit) aqueous solutions of 1 are treated with very dilute solutions of 2 or 3, there is a strong quenching of the J-aggregate (13) We thank Dr. Thomas Penner for providing us a sample of this dye.
Figure 2. Absorption and emission spectra of dye polymer 1 upon sequential addition of cyanine dye monomer 3: solid line, polymer 1 absorption; dotted line, polymer 1 emission with no dye 3 added; dot-dash and dashed lines, emission spectra recorded on sequential addition of 3.
fluorescence of the cyanine dye. For both quenchers the decrease in fluorescence with addition of quencher follows a linear Stern-Volmer relationship and extremely large Ksv values are obtained (Table 1). For the anthraquinone quencher 2, which is anticipated to quench by electron transfer, there is no new fluorescence as a consequence of the quenching and no change in either the J-aggregate absorption or emission. While the quenching is assumed to be entirely “static” due to the formation of an association complex between a single repeat unit of 1 and 2, any spectral change occurring as a result of this complex formation cannot be detected at the low quencher levels employed. In pure water, 50% quenching of a solution of 1 (2 × 10-6 M in repeat units) is observed at a concentration of 2 of 1.5 × 10-8 M. This corresponds to 1 quencher per 133 repeat units; since the polymer has an average of 250 repeat units per chain this means that the polymer is effectively quenched at a level of one to four quenchers per polymer chain. Similar results are obtained when polymer 1 is examined in mixtures of 50% water/50% dimethyl sulfoxide; the absorption and emission spectra of the polymer are similar to those obtained in water as is the Ksv and quencher level at which the polymer is quenched (Table 1). The latter result is significant in terms of comparison to the conjugated polyelectrolyte 4 studied previously;1 for this compound there are significant changes in both the absorption/emission profile with a similar change in solvent as well as a large decrease in the sensitivity to quenching.14 “Superquenching” of 1 by 2 persists even in the presence of high electrolyte concentration; however, as might be anticipated, the magnitude of the quenching is somewhat attenuated. The anionic cyanine dye 3 also quenches the fluorescence of 1 at very low concentrations (Ksv in water ∼4 × 107 M-1); however in this case there is appearance of a relatively weak new fluorescence at 630 nm (Figure 2) which corresponds to neither that of 1 nor 3 but is at slightly longer wavelengths than the “monomer” fluorescence of 3 which has a maximum at 612 nm.15 The emission of 1 is quenched by more than 50% when the ratio of 3 to polymer repeat unit is 1 to 50. Since cyanine dye 3 appears more likely to be an energy acceptor for the repeat unit (14) Jones, R. M. Unpublished results. (15) It is not clear as to the origin of the small differences in SternVolmer quenching constants between 2 and 3 (for 1). While 3 might be expected to quench at longer range than 2 (energy transfer for 3 vs electron transfer for 2), the greater net charge on 2 may result in its having a stronger association constant with 1.
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of 1 than an electron acceptor, it appears reasonable to assign the new fluorescence to a “complexed” form of 3 that is selectively activated by an energy transfer process from photoexcited 1. Since 1 is a polyelectrolyte, only sparingly soluble in water, it is reasonable that exposure of solutions of 1 to anionic surfaces, beads, or particle suspensions can result in deposition of the polymer onto the surface. This has been previously reported for 1 for solid surfaces decorated with Laponite clays.10 For these surfaces both the corresponding monomer cyanine dye and polymer 1 give J-aggregates having absorption and fluorescence almost identical to those spectra shown in Figure 1. Interestingly, when 1 is treated with an aqueous suspension of the same Laponite clay, the aggregate absorption is significantly blue-shifted and broadened and there is a measurable broadening of the aggregate fluorescence. However, when solutions of 1 whose fluorescence is partially quenched by 2 are transferred to the same clay suspensions under high loading conditions, the fluorescence of the adsorbed cyanine aggregate exhibits significantly greater quenching
than is observed for the corresponding solutions. The enhanced “superquenching” observed in this situation (Ksv increases from 7.7 × 107 to 1.03 × 108 M-1) is initially surprising since it might be expected that some quencher would be excluded by repulsion from the clay surfaces or that the “imperfect” J-aggregates transferred (inferred by the blue-shifted absorption and emission spectra on clay) might be less efficient in terms of energy migration or exciton delocalization within the polymer. There may exist some precedent for this in earlier studies with Langmuir-Blodgett films of amphiphilic cyanine dyes by Kuhn, Mo¨bius, and co-workers.16-18 In one of the most impressive reported examples of “superquenching” in a supramolecular assembly, these authors observed that doping of a very small amount of an energy acceptor cyanine in a single layer otherwise composed of a donor (16) Mo¨bius, D.; Kuhn, H. J. Appl. Phys. 1988, 64, 5138. (17) Kuhn, H.; Foersterling, H.-D. Principles of Physical Chemistry; Wiley: Chichester, 2000; pp 805-810. (18) Kuhn, H.; Kuhn, C. In J-Aggregates; Kobayashi, T., Ed.; World Scientific: Singapore, 1996.
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cyanine in its J-aggregated form resulted in quenching of the aggregate fluorescence and appearance of the acceptor cyanine fluorescence (similar to the quenching of 1 by 3). The unique photophysical and chemical properties of the J-aggregated polymer offer some considerable advantages in applications of the superquenching. In contrast to the conjugated polymer 4, used in previous studies, which shows broad absorption and fluorescence, sensitivity to a wide variety of additives, and susceptibility to a variety of nonradiative decay paths, the J-aggregated polymers offer very narrow absorption and emission, a great variety of possible polymers both in terms of the cyanine dye and molecular weight of the polymer, and a relatively lower sensitivity to nonspecific effects induced by the environment. We have carried out some studies with the J-aggregated polymer 1 in biosensing via fluorescence quenchingunquenching experiments. Upon addition of small amounts of the fluorescent QTL conjugate, 5 we observed quenching of the emission from 1 concurrently with the sensitized emission of 5. When small amounts of avidin are added to solutions of 1 quenched by 5, there is very little recovery of the fluorescence of 1, despite evidence that the biotin ligand of 5 associates with the protein. In this case it seems clear that the avidin associates with the polymer and that binding of the biotin of the QTL conjugate does not suffice to remove the quencher from the vicinity of the polymer. Experiments with only 1 and avidin indicate that the polymer and protein associate strongly. However an experiment conducted with solutions of polymer 1 separated from solutions containing the avidin-QTL conjugate 6 by the cellulose (NMWCO ) 30 kDa) membrane of a Microcon YM 30 ultrafiltration device indicates that neither the polymer nor the protein/conjugate complex can pass through the membrane, thus affording an alternate application for using the polymer superquenching in a homogeneous competitive bioassay. A competitive assay was demonstrated by first complexing the anthraquinone-biotin QTL (6) with avidin in a molar ratio 0.8:1 (6:avidin). Since each avidin has four biotin-binding sites, this substoichiometric ratio ensures full binding of the QTL. As expected, upon centrifugation, this complex does not pass through the membrane, and no quenching is observed. No quenching is observed also if only biotin is present. However, upon adding biotin to the QTL-avidin solution in a 4:1 ratio (biotin:avidin), it is expected that
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Figure 3. A competition assay with polymer 1 conducted in YM-30 ultrafiltration vials (NMWCO ) 30 kDa). Prior to centrifugation the vial compositions were as follows: (1) lower solution for each vial, 0.1 µM 1; (2) upper solution (retentates), 0.5 µM D-biotin (dashed line), 0.8 µM AQS-biotin (6) + 1.0 µM avidin (dotted line), and 0.8 µM AQS-biotin (6) + 1.0 µM avidin with 0.5 µM D-biotin added (solid line). The fluorescence of 1 is shown for each vial after incubation and centrifugation (see Experimental Section for details).
competition of biotin with 6 for the avidin binding sites will result in release of the QTL and subsequent quenching. This is observed, as shown in Figure 3. In summary, we have shown that the phenomenon of superquenching first discovered for conjugated polymers with small molecule electron transfer quenchers also extends to polymers in which formally nonconjugated chromophores associate through J-aggregation. For the J-aggregate polymers we have shown that the sensitivity to “superquenching” may be extended to quenchers that interact via energy transfer or fluorescent exciplex formation. The use of a quencher-tether-ligand bioconjugate as the quencher affords the use of the highly fluorescent J-aggregate polymers as the basis for a highly selective, sensitive, simple, and rapid biosensor. Acknowledgment. This work was supported by the Defense Advanced Research Projects Agency under Contract MDA972-00-C-006. LA001745L
