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2007, 111, 14607-14611 Published on Web 09/20/2007
Zinc Sensing via Enhancement of Two-Photon Excited Fluorescence Ajit Bhaskar,‡ Guda Ramakrishna,† Robert J. Twieg,§ and Theodore Goodson, III*,†,‡ Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109, Department of Macromolecular Science and Engineering, UniVersity of Michigan, Ann Arbor, Michigan 48109, and Department of Chemistry, Kent State UniVersity, Kent, Ohio 44242 ReceiVed: August 2, 2007; In Final Form: August 24, 2007
A branched chromophore tris[p-(4-pyridylethynyl)phenyl]amine (TPPA) with pyridine terminal groups is investigated for its ability to sense small quantities of Zinc ions via a two-photon excited fluorescence (TPEF) enhancement mechanism. Changes in the linear and nonlinear optical properties upon coordination with Zn2+ have been studied using steady-state measurements, Two-photon absorption and femtosecond transient absorption spectroscopy. An order of magnitude increase in the TPEF signal of the Zn-bound chromophore was observed, thereby rendering our chromophore a “turn-on” sensor for Zn2+. The molecule was highly selective toward Zn2+ and was able to detect Zn2+ at the ppm level. Transient absorption measurements revealed the excited-state dynamics and elucidated the reasons for the observed behavior of the TPPA chromophore. This study provides a guideline along which organic chromophores could be developed for “turn-on” metal ion sensing using multiphoton excitation.
1. Introduction Two-photon absorption (TPA) is an example of a third-order nonlinear optical process. It offers distinct advantages over onephoton processes such as quadratic dependence of two-photon excited fluorescence (TPEF) intensity on input power, increased penetration depth, and reduced scattering among many others, which renders TPA attractive for several applications.1,2 One such rapidly emerging application lies in metal ion sensing using near infrared wavelengths.3 In this paper, we report a system that shows enhancement in two-photon excited fluorescence (TPEF) upon formation of a complex with zinc ions. There are reports on systems involving zinc ions with interesting TPA behavior. Righetto et al. investigated branched chromophores with pyridine groups and reported a decrease in the TPA cross section upon coordination with zinc ions.4 The authors explained this observation on the basis of a two-state model, suggesting that coordination with zinc resulted in a species with its ground state closer to the “cyanine” form, which in turn would cause a decrease in the TPA cross section.4 Fabbrini et al. have reported a chromophore that shows an increase in TPA cross section upon binding with zinc using nanosecond pulse nonlinear absorption method. However, no information on fluorescence behavior was reported.5 Bozio et al. have developed a zinc chemosensor based on a sharp decrease in TPEF response as zinc ions were added.6 The authors attributed this to a change in the tetramethylcyclen moiety from donor to acceptor upon coordination with zinc ions. Ahn et al. have also observed a similar trend.7 Das et al. have reported a Schiff’s base type ligand, which showed enhancement * Corresponding author. E-mail:
[email protected]. † Department of Chemistry, University of Michigan. ‡ Department of Macromolecular Science and Engineering, University of Michigan. § Kent State University.
10.1021/jp076208p CCC: $37.00
Figure 1. Structure of TPPA chromophore.
of the TPA cross section as it coordinated with zinc.8 Liu et al. performed a theoretical study on multipyridyl ligand chromophores and predicted an increase in the TPA cross sections upon coordination with zinc due to the spiroconjugation effect.9 However, little information was provided regarding the TPEF behavior. To the best of our knowledge, a system that shows substantial increases in both the TPEF signal and TPA cross section upon coordination with zinc has not been reported. In this study, we report a molecule (structure shown in Figure 1) that can detect zinc at the parts per million (ppm) level. It forms a complex with Zn2+ and subsequently shows an order of magnitude increase in the net TPEF signal (ηδ) and significant improvement in TPA cross section (δ) in the near IR excitation region, in spite of decrease in fluorescence quantum yield. In this article, we report a TP fluorescence-based sensor that shows significant improvement in ηδ as well as δ upon coordination only with Zn2+. It is highly sensitive and selective toward zinc ions. Our sensor does not yield a positive TPEF response to Cd2+, which has been a challenge in the selective detection of zinc ions. We also investigate the effect of branch length on TPEF behavior and provide the mechanism of selectivity and sensitivity of the chromophore toward Zn2+. © 2007 American Chemical Society
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Figure 2. (A) Absorption spectra for TPPA (black curve). (B) Fluorescence emission spectra for TPPA and TPPA-Zn. 335-nm excitation was used to obtain the emission spectra.
The present work, therefore, provides a guideline along which novel materials for multiphoton excited “turn on” metal ion sensing that could be potentially used for TP imaging, and other applications could be designed and developed. 2. Experimental Section a. Synthesis of the Chromophores. The synthesis of the main chromophore under investigation, tris[p-(4-pyridylethynyl)phenyl]amine (addressed as TPPA from here onward) has been reported previously.10 b. Steady-State Measurements. All compounds were used as received without any further purification. Unless otherwise stated, all measurements were carried out in tetrahydrofuran (THF) of spectrophotometric grade, purchased from SigmaAldrich. c. Two-Photon Absorption Cross Section Measurements. In order to measure the two photon absorption cross sections, we followed the TPEF method.11 The details of the lasers and optical setup have been described elsewhere.10 A 10-4 M Coumarin 307 (Acros Organics) solution in methanol was used as the reference over the 700-830 nm range. p-Bis(o-methylstyryl) benzene or MSB (Sigma-Aldrich) was used as the reference over the 550-695 nm range. Quadratic dependence of TPEF intensity on input intensity was ensured at every wavelength. d. Ultrafast Transient Absorption Measurements. Femtosecond transient absorption investigations have been carried out using ultrafast pump-probe spectrometer detecting in the visible region. The details of this setup have been published elsewhere.10 The pump beams used in the present investigation were 375 nm for TPPA and 430 nm for the TPPA-Zn2+ complex. 3. Results and Discussion 3.1. Steady-State Properties. The optical absorption and fluorescence results for TPPA-Zn2+ are shown in Figure 2. To 800 µL of 7.05 × 10-7 M TPPA, zinc chloride in THF was added in a stepwise fashion (Supporting Information). Different concentrations of ZnCl2 were used, ranging from 0.5 to 25 mM. As a control, identical volumes of pure THF were added in a separate sample cell. No change in absorption or emission behavior was observed except for decrease in peak intensity values. A bathochromic shift in both absorption and emission spectra upon coordination with Zn2+ was observed, as indicated in Figure 2. The broadening and red shift of the absorption
spectrum for TPPA-Zn2+ suggest the formation of a partial charge-transfer-type complex (between the pyridine end group and Zn2+). The quantum yield of the coordinated complex was determined to be 0.27, whereas it was 0.58 for TPPA alone. The data presented in Figure 2 was used for determining the binding constant (Supporting Information). Following a known procedure,12 a 1:1 complex proved to be the best fit for the data obtained. The value of K1 was found to be 7200 M-1, which is comparable to some of the previously reported molecules in the literature.12,13 Besides ZnCl2, other zinc salts such as zinc acetate and zinc cyanide were added to TPPA and the formation of complex was still observed, thereby precluding any spurious effects due to anions. We also added ZnCl2 in THF to a previously reported chromophore tris[4-(3′, 5′-di-tert-butyldistyryl benzenyl) phenyl] amine N(DSB)3,10 which has the same tribranched nitrogen core as TPPA, but with alkene π-bridges and a tert-butyl phenyl end group. We did not observe any change in the absorption or emission characteristics. This affirms that coordination occurs between Zn2+ and the pyridine end groups and that the core nitrogen atom does not participate in the complexation process. The use of pyridine group to coordinate with zinc ions has been reported in the literature.4-9 However, most reports concentrate on the ligand in a bipyridyl or a multipyridyl configuration. In the present study, the TPPA chromophore has pyridine units where the separation between the nitrogen atoms is larger than the commonly reported multipyridyl chromophores used for coordination with zinc ions in the literature.4-9 3.2. TPA Measurements. TPEF and TPA cross-section measurements were carried out using the same solutions that were used for the determination of binding constant and quantum yields. The one- and two-photon excited fluorescence spectra for TPPA and TPPA-Zn2+ are shown in Figure 3. The TPEF spectra were measured at identical incident photon flux. Because the dye concentration was identical in both TPPA and TPPAZn2+, it can be concluded that TPPA-Zn2+ shows ∼11.5 fold increase in the TPEF signal upon comparison of the peak TPEF intensities of TPPA-Zn2+ and TPPA. However, if the TPEF at 550 nm is monitored, then we observed 2 orders of magnitude enhancement in the TPEF intensity. This is in contrast to steadystate fluorescence behavior where coordination with zinc decreases the fluorescence intensity at the emission maximum. Hence, coordination with zinc not only results in a significant enhancement of TPA cross-section but also increases the TPEF signal. We have also carried out measurements with branched chromophores with structures similar to that of TPPA. These
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Figure 3. One- and two-photon excited fluorescence spectra for different chromophores and chromophore-Zn2+ complexes. Steady-state measurements were performed at 330-nm excitation, whereas TPEF spectra were obtained at 804 nm.
TABLE 1: Selectivity of TPPA toward Zn2+ metal (oxidation number) ηδ 800 nm (GM)
Zn Cd Au Au Cu Ni Fe Fe (+2) (+2) (+1) (+3) (+2) (+2) (+3) (+2) 193
9.7
7.3
5.3
0.1
0.3
0.8
0.5
other chromophores had longer branch lengths and/or alkene π-linkages instead of alkyne linkages (as with TPPA) or both. Similar bathochromic shifts in both absorption and emission spectra for all of the chromophores were observed. However, none of the other chromophores showed enhancement of the TPEF signal (Supporting Information). Hence, the mere presence of pyridine groups does not explain the observed TPEF enhancement for TPPA. More detailed insight into the steadystate and excited-state properties is desired in order to elucidate the observed TPEF trends. Investigations were also carried out on other metal ions such as Cd2+, Au (both Au3+ and Au+), Ni2+, Cu2+, Cu+, Ag+, and Co2+. All of them showed an enhanced δ value, but the TPEF signal (ηδ) dropped dramatically. The results are summarized in Table 1. Other metal ions such as Na+, K+, Ca2+, and Mg2+ did not alter the steady-state as well as TPEF behavior. Hence, only Zn2+ shows an enhancement in both δ and ηδ, suggesting that TPPA is a selective, two-photon “turn-on” sensor for Zn2+. The TPEF signal at 550 nm as a function of excitation wavelength for TPPA and TPPA-Zn2+ is presented in Figure 4A. It can be observed that for the entire wavelength range, the TPEF signal for TPPA-Zn2+ is substantially higher than that for TPPA. In fact, beyond 800 nm, an order of magnitude enhancement in both ηδ and δ was observed for TPPA-Zn2+. To measure the sensitivity of TPPA toward Zn2+, we added small volumes of ZnCl2 stock solutions to 1 mL of 4.23 × 10-7 M TPPA. The results are shown in Figure 4B. From the linear fit shown, it is evident that in order to increase the TPEF signal threefold, the amount of Zn2+ required is 12.8 µM. This translates into a detection limit of 0.8 mg/L or 0.8 ppm. This sensitivity is comparable with that of some of the zinc sensors reported in the literature.14,15 Sensors that claim to detect Zn2+ in the nanomolar and femtomolar regime based have been reported.16,17 However, the authors have not addressed the selectivity of those sensors toward zinc. Table 1 further demonstrates the selectivity of TPPA towards zinc (II) over Cd(II), which is one of the challenges encountered while designing zinc sensors.18,19 Thus, the present molecule TPPA serves as a highly selective and sensitive multiphoton probe for Zn2+.
3.3. Femtosecond Transient Absorption Measurements. To understand the mechanism behind the observations from steady-state and TPEF measurements, we have investigated the excited-state dynamics of TPPA with and without metal ions using ultrafast pump-probe spectroscopy. Figure 5A and B shows the excited-state absorption (ESA) spectra of the FranckCondon (FC) state at 100 fs and final emitting state at 20 ps for TPPA in THF and TPPA with Zn2+, respectively. It can be observed from Figure 5A that the FC state’s ESA spectrum is unchanged upon complexation with Zn2+. However, the steadystate results show that the acceptor strength of pyridine is increased with metal ion complexation. This result suggests that there might a possible breaking or significant elongation of the Zn2+-pyridine bond upon photoexcitation, leading to a large change in dipole moment (∆µge). To relate this to enhancement of TPA cross section, we utilized a sum-over-states (SOS) expression for calculating the TPA cross sections for noncentrosymmetric molecules,19 which is shown in eq 1. We concentrate on the lower-energy absorption peak of the TPPA chromophore. In this case, there is no contribution from the excited-state transition dipole moment (Mee′). There is some bathochromic shift of the absorption spectrum upon addition of Zn2+, but it does not explain the order of magnitude enhancement of TPEF that we have observed. Steady-state measurements with Zn2+ suggest that there is not a significant change in the ground-state transition dipole moment (Mge). The bathochromic shift in both absorption and emission maxima suggest a ground-state complex between TPPA and Zn2+. The present transient absorption measurements suggest that the possible reason for large increase in TPA cross section arises from ∆µge.
δgfeR
Mge2∆µge2
( ) Ege2 4
(1)
The suggestion for possible bond TPPA-Zn2+ bond cleavage arises from a previously published report on hydrogen-bonded Coumarin-amine system.20 However, the FC state appears to reform the bond with Zn2+ with a time constant of 400 fs to give TPPA-Zn2+ which is followed by solvation to give an ESA maximum at 540 nm with a shoulder at 500 nm and stimulated emission around 610 nm (Figure 5B). These transient features are ascribed to that of singlet-singlet absorption of TPPA-Zn2+ intramolecular charge transfer (ICT) state. Other chromophores also show bond elongation but not as pronounced as TPPA (Supporting information). Also, there is additional formation regarding a non-emissive intramolecular charge-
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Figure 4. (A) TPEF signal values for TPPA and TPPA-Zn2+ at different excitation wavelengths. The values listed are for 550-nm detection for both TPPA and TPPA-Zn2+. (B) Measurement of sensitivity of TPPA toward Zn2+. 800-nm excitation was used for measuring TPEF signals.
Figure 5. Excited-state absorption spectra of FC state at 150 fs of TPPA and TPPA-Zn2+ (A) and at 20 ps of TPPA and TPPA-Zn2+ (B) after femtosecond laser excitation (fwhm ∼130 fs).
transfer state (with a lifetime of 2-3 ps), which is a solvent conformationally relaxed state for other dye molecules,21 and this is the probable reason behind the low fluorescence quantum yields observed for other dye molecules upon complexation with Zn2+. Thus, the present transient results were able to provide possible mechanism for the enhancement of the TPA cross section of TPPA in the presence of Zn2+. The question that also needs to be addressed here is why is the fluorescence quantum yield of TPPA with Zn2+ is higher compared to the results with other metal ions? The analysis of the kinetic traces of TPPA with Zn2+ and Au3+ (Supporting Information) shows that the lifetime of the emitting state of TPPA-Zn2+ has a lifetime greater than 2 ns, whereas that of TPPA-Au3+ is only 20 ps (Supporting Information). The fall in radiative lifetimes is the main reason behind low quantum yield, and thereby a lower TPEF signal is observed for TPPA in the presence of other metals. Transient measurements corroborate the findings from TPEF measurements and elucidate the selectivity and sensitivity of the TPPA dye for Zn2+ alone and not for other metal ions. 4. Conclusions A TPEF turn-on sensor for selective and sensitive detection of Zn2+ has been developed. Significant enhancement in both the TPEF signal as well as the TPA cross section was observed
over a wide range of wavelengths. Increase in the acceptor strength and larger change in dipole moment are the suggested reasons behind the enhancement of the TPA cross section upon addition of Zn2+. Enhancement of the TPEF signal with Zn2+ is observed for only TPPA and not for other structurally similar branched molecules with pyridine as the end group. The presence of a non-emissive state in the excited-state dynamics is suggested as the mechanism behind the lower TPEF signal. Because the TPEF signal of TPPA can be enhanced only by Zn2+, it could find applications in several biological systems such as amyloids and prion peptides. Their interaction with zinc and possible role in neurological disorders has been a subject of significant investigation,22,23 which can be well studied with the present TPPA and can be imaged using two-photon excitation. PrP106-126 is known to have 7 orders of magnitude larger affinity for Cu2+ than Zn2+.24 With the present approach, one could design a chromophore that shows TPEF enhancement specifically upon coordinating with Zn2+ in order to gain further insight into the role of zinc in Alzheimer’s disease. Supporting Information Available: TPA measurements on TPPA, T-233, T-161, and T-119, detailed transient absorption results for TPPA, T-233, T-161, and T-119 with and without Zn2+ and other metal ions such as Au3+, and determination of binding constant. This material is available free of charge via the Internet at http://pubs.acs.org.
Letters References and Notes (1) Cumpston, B. H.; Ananthavel, S. P.; Barlow, S.; Dyer, D. L.; Ehrlich, J. E.; Erskine, L. L., Heikal, A. A.; Kuebler, S. M.; Lee, I.-Y. S.; McCord-Maughon, D.; Qin, J.; Ro¨ckel, H; Rumi, M.; Wu, X.-L.; Marder, S. R.; Perry, J. W. Nature 1999, 398, 51. (2) Kawata, S.; Sun, H.-B.; Tanaka, T.; Takada, K. Nature 2001, 412, 697. (3) Huang, F.; Tian, Y.; Ching-Yi, C.; Young, C. A.; Jen, A. K. J. Phys. Chem. C 2007, 111, 10673. (4) Righetto, S.; Rondena, S.; Locatelli, D.; Roberto, D.; Tessore, F.; Ugo, R.; Quici, S.; Korystov, D.; Srdanov, V. I. J. Mater. Chem. 2006, 16, 1439. (5) Fabbrni, G.; Ricco´, R.; Menna, E.; Maggini, M.; Amendola, V.; Garbin, M.; Villano, M.; Meneghetti, M. Phys. Chem. Chem. Phys. 2007, 9, 616. (6) Bozio, R.; Cecchetto, E.; Fabbrini, G.; Ferrante, C.; Maggini, M.; Menna, E.; Pedron, D.; Ricco`, R.; Signorini, R.; Zerbetto, M. J. Phys. Chem. A 2006, 110, 6459. (7) Ahn, H. C.; Yang, S. K.; Kim, H. M.; Li, S.; jeon, S.-J.; Cho, B. R. Chem. Phys. Lett. 2005, 410, 312. (8) Das, S.; Nag, A.; Goswami, D.; Bharadwaj, P. K. J. Am. Chem. Soc. 2006, 128, 402. (9) Liu, X.; Feng, J.; Ren, A.; Cheng, H.; Zhou, A. J. Chem. Phys. 2004, 120, 11493. (10) Bhaskar, A.; Ramakrishna, G.; Lu, Z.; Twieg, R. J.; Hales, J. M.; Hagan, D. J.; Van Stryland, E.; Goodson, T., III J. Am. Chem. Soc. 2006, 128, 11840. (11) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481.
J. Phys. Chem. C, Vol. 111, No. 40, 2007 14611 (12) Pond, S. J.; Tsutsumi, O.; Rumi, R.; Kwon, O.; Zojer, E.; Bredas, J.-L.; Marder, S. R.; Perry, J. W. J. Am. Chem. Soc. 2004, 126, 9291. (13) Marcotte, R.; Plaza, P.; Lavabre, D.; Fery-Forgues, S.; Martin, M. M. J. Phys. Chem. A 2003, 107, 2394. (14) Chang, C. J.; Nolan, E. M.; Jaworski, J.; Okamoto, K.-I.; Hayashi, Y.; Sheng, M.; Lippard, S. J. Inorg. Chem. 2004, 43, 6774. (15) Nolan, E. M.; Burdette, S. C.; Harvey, J. H.; Hilderbrand, S. A.; Lippard, S. J. Inorg. Chem. 2004, 43, 2624. (16) Bontidean, I.; Berggren, C.; Johansson, C.; Cso¨regi, E.; Mattiasson, B.; Lloyd, J. R.; Jakeman, K. J.; Brown, N. L. Anal. Chem. 1998, 70, 4162. (17) McCarroll, M. E.; Shi, Y.; Harris, S.; Puli, S.; Kimaru, I.; Xu, R.; Wang, L.; Dyer, D. J. J. Phys. Chem. B 2006, 110, 22991. (18) Wang, J.; Xiao, Y.; Zhang, Z.; Qian, X.; Yang, Y.; Xu, Q. J. Mater. Chem. 2005, 15, 2836. (19) Beljonne, D.; Wensellers, W.; Zojer, E.; Shuai, Z.; Vogel, H.; Pond, S. J.; Perry, J. W.; Bredas, J.-L. AdV. Funct. Mater. 2002, 12, 631. (20) Palit, D. K.; Zhang, T.; Kumazaki, S.; Yoshihara, K. J. Phys. Chem. A 2003, 107, 10798. (21) Ramakrishna, G.; Bhaskar, A.; Goodson, T., III J. Phys. Chem. B 2006, 110, 20872. (22) Grasso, D.; Milardo, D.; La Rosa, C.; Rizzarelli, E. Chem. Comm. 2004, 246. (23) Eliza-Diana, C.; Meckmouche, Y.; Faller, P. Chem.sEur. J. 2005, 11, 903. (24) Gaggelli, E.; Bernardi, F.; Molteni, E.; Pogni, R.; Valensin, D.; Valensin, G.; Remelli, M.; Luczkowski, L.; Kozlowski, H. J. Am. Chem. Soc. 2005, 127, 996.
