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Cite This: J. Am. Chem. Soc. 2018, 140, 10975−10979
Excitation- and Emission-Wavelength-Based Multiplex Spectroscopy Using Red-Absorbing Near-Infrared-Emitting Lanthanide Complexes Ruisheng Xiong,† Dimitrije Mara,‡ Jing Liu,‡ Rik Van Deun,‡ and K. Eszter Borbas*,† †
Department of Chemistry, Ångström Laboratory, Uppsala University, Lägerhyddsvägen 1, 75120 Uppsala, Sweden L −Luminescent Lanthanide Lab, Department of Chemistry, Ghent University, Krijgslaan 281, Building S3, B-9000 Gent, Belgium
‡ 3
J. Am. Chem. Soc. 2018.140:10975-10979. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/31/18. For personal use only.
S Supporting Information *
ABSTRACT: Multiplex imaging in the red and nearinfrared (NIR) should be an enabling tool for the realtime investigation of biological systems. Currently available emitters have short luminescent lifetimes, broad absorption and emission bands, and small Stokes shifts, which limits multiplexing in this region to two colors. NIR-emitting luminescent lanthanide (Ln) complexes carrying hydroporphyrin (chlorin) sensitizing antennae are excitable in the red through the narrow, intense and tunable chlorin absorptions. Both emission- and excitation-based multiplexing are possible, the former by exciting the same antenna appended to different Lns, the latter by attaching different chlorins with nonoverlapping absorptions to the same Ln. The combination of excitation and emission spectroscopies allows for the straightforward differentiation of up to four different complexes. ultiplex fluorescence imaging offers information at a level of detail that is beyond the reach of single fluorophores. These tools are particularly valuable for studying complex systems, e.g., in fundamental cell biology or in cancer diagnostics.1−7 Small-molecule organic fluorophores and fluorescent proteins are of limited use in multiplexing due to the broadness of their absorption and emission bands, which results in spectral crosstalk and complicated data interpretation. Emitters based on luminescent lanthanide complexes [Ln(III)] overcome this limitation, and enable both wavelength-based8−10 and lifetime-based6,11 multiplexing. The former is by virtue of the sharp and mostly nonoverlapping, metalspecific Ln emission peaks. The latter technique is underpinned by the variety of excited state lifetimes found in Ln(III) complexes, which can cover over 6 orders of magnitude, from hundreds of ns to several ms.12 Higher-order multiplexing, i.e., the combination of temporal and spectral signal separation, is also possible: a long-lived Tb donor and two cyanine acceptors placed at different distances have enabled the differentiation of up to 4 targets.13 The Laporte-forbidden Ln(III) luminescence can be sensitized using light-harvesting chromophores (“antennae”). For biological applications, the near-infrared (NIR) emitting Lns, Yb(III) and Nd(III), are preferred to minimize tissue damage and maximize signal penetration. Here, we show that the multiplexing capacity of NIR Ln(III) can be greatly expanded by combining them with red-absorbing hydroporphyrin (chlorin, Figure 1) light harvesting antennae, which allow for excitation-wavelength-based discrimination in
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© 2018 American Chemical Society
Figure 1. (a) Comparing the multiplexing abilities of organic fluorophores and of chlorin−Ln dyads. The Cy5/Cy7 absorptions and emissions cover the 500−800 nm range, and they substantially overlap. Two judiciously chosen chlorins combined with two different Lns enable the separate addressing of two distinct excitation and two distinct emission channels. (b) Prepared Ln−chlorin dyads.
addition to emission-based multiplexing. Up to 4 luminescent species could be readily identified. Chlorins have intense and narrow absorptions (Qy-bands) in the red. This is beneficial for the optical imaging of biological materials due to the nondestructiveness of such low-energy Received: July 18, 2018 Published: August 18, 2018 10975
DOI: 10.1021/jacs.8b07609 J. Am. Chem. Soc. 2018, 140, 10975−10979
Communication
Journal of the American Chemical Society light, and the better transparency of biological structures at long wavelengths. The position and intensity of this band is tunable by peripheral substitution and macrocycle metalation. For the types of chlorins used here (Figure 1b) ε(Qy) ∼ 39 500−90 300 M−1·cm−1 are typical.14,15 We hypothesized that chlorin antennae would provide access to red-absorbing, NIR-emitting Ln complexes with nonoverlapping excitation bands. Though Yb complexes with chlorin antennae have been reported,16−18 most were not amenable to Q-band excitation, and the lack of excitation spectra left the sensitization by the chlorin unproven. Recently, Zhang and co-workers have developed Yb emitters based on perfluorinated porpholactones and azlactols.19,20 These elegant systems allowed for efficient red excitation. However, no Nd analogues were reported, and the Q-bands are also weaker and broader than those in the chlorins,15 which would make positional tuning and excitationbased multiplexing challenging. To overcome the challenges encountered with previous chlorin−Ln dyads, we designed 3:1 homoleptic and 1:2:1 heteroleptic complexes based on a dipicolinic acid21 (dpa) framework (Figure 1). Free base and Pd chelate chlorins were appended to the 4-position of the dpa through either the 3- or the 15-position of the chlorin to yield a series of antennae with a range of Q-band absorption maxima (vide infra). The short Ln-antenna distances should enable efficient energy transfer. Quenching O−H, N−H and C−H oscillators22 are excluded from the first coordination sphere. The 3 antennae in the 3:1 complexes were expected to increase complex brightness. Chlorins were prepared using modifications of the Lindsey synthesis (see Supporting Information).23−25 Peripheral substituents were introduced by Pd-catalyzed cross-couplings using the appropriate bromochlorin precursors.26,27 Ln complexation was performed by titrating a known amount of LnCl3 or [Ln(dpa)2]− (ref 28) with ChlxM2‑. The ligands were fully characterized using 1H and 13C NMR analysis, HR-ESI-MS and UV−vis absorption and emission spectroscopy. The identities of the complexes were supported by luminescence emission and UV−vis absorption spectroscopies (vide infra). The UV−vis absorption spectra of the chlorins are shown in Figure 2a. Pd-chelates had ∼50 nm blue-shifted Qy-bands compared to the analogous free base compounds. The differences in the Soret band positions were negligible. The Qy-band absorptions of free base chlorins and Pd chelates were well separated. The influence of the dpa-substitution was smaller than that of the metalation. 15-linked antennae had blueshifted Qy-bands (by ∼13-21 nm) compared to 3-linked ones. These observations are in line with previous reports on the effect of metalation and aromatic peripheral substitution.18,29 Importantly, these antennae can be combined pairwise to enable selective excitation of one in the presence of the other (e.g., Chl3Pd or Chl15Pd with Chl3FB or Chl15FB). Apart from spectral tuning, palladation also promotes intersystem crossing, thus facilitating triplet-mediated sensitization pathways. Chlorin excitation afforded Ln-centered emission in every Ln complex (Figures S18−S55), the Ln excitation spectra were similar to the antenna absorptions (Figures 2b, S1−S17), confirming that Ln emission was sensitized by the chlorins. Crucially, the Qy-bands were clearly visible in the excitation spectra (at λmax = 600 nm for Chl3Pd, λmax = 590 nm for Chl15Pd, λmax = 645 nm for Chl3FB, and λmax = 637 nm for Chl15FB). This unambiguously establishes that these tunable red features are suitable for the sensitization of NIR Ln
Figure 2. (a) Chlorin absorption spectra in MeOH, [Chlx] = 0.5 mM. (b) Ln excitation spectra of complexes carrying Chl15Pd antennae in THF/water. (c and d) Excitation spectra of Nd (c) and Yb (d) complexes carrying the same chlorin antennae in different architectures. [Chlx] = 27 μM ([complex] = 9 μM), λem = 980 nm for Yb, and 1060 nm for Nd. The uncomplexed ligands are not soluble in aqueous mixtures, hence the choice of MeOH for recording the absorption spectra. 10976
DOI: 10.1021/jacs.8b07609 J. Am. Chem. Soc. 2018, 140, 10975−10979
Communication
Journal of the American Chemical Society emission; λex > 650 nm were available for both Yb and Nd complexes. Notably, fluorescence microscopy usually relies on near UV or blue-light excitation. While two-photon excitation is possible with red/NIR light, it requires expensive instrumentation, and the development of new emitters can be challenging.30−32 Excitation maxima were only dependent on the chlorin and not on the Ln (Figure 2b). The antennae in heteroleptic complexes seemed to be more efficient sensitizers than in the homoleptic ones, despite the latter complexes having 3 times as many antennae (Figure 2c,d). This may be due to interaction of the chlorins in the 3:1 complexes quenching the antenna excited states.33 The excitation wavelengths used here are the most red-shifted ones reported to date, exceeding the previously most red-shifted excitation wavelengths (600 nm)19 by >50 nm. The sensitized Nd emission spectra showed two peaks in the observed region, assigned to the 4F3/2→4IJ (J = 9/2, 11/2; 890, 1064 nm, respectively) transitions. The 890 nm band overlapped with an emissive contaminant (or an instrumental artifact), which was also observed in some of the Yb spectra. Therefore, we relied on the 1064 nm peak for detecting Nd and the 980 nm peak for Yb. The latter corresponds to the 2 F2/5→2F2/7 transition. With these data in hand, we evaluated the potential of our complexes for excitation- and emission-based multiplexing. A mixture of Nd and Yb complexes that carried Chl15Pd antennae were excited at the chlorin Q-band (λex = 590 nm), which afforded both Nd and Yb emissions (Figure 3a).
We could distinguish two Yb complexes carrying two different antennae (Chl15FB and Chl15Pd) due to the differences in their excitation spectra (Figure 3b). A combination of the Yb complexes of the free base and the Pd-chelates of Chl15 had an excitation spectrum displaying two distinct, well-separated excitation peaks in the red. The peaks were located at the same wavelengths as those of the individual components (Figure 3b). These results show that both excitation- and emission-based differentiation is possible in mixtures of Ln complexes, and that chlorin metalation (palladation) provides a sufficiently large shift of the Q-band to enable interference-free excitation of either the free base or the Pd chlorin in the presence of the other. These Ln complexes were substantially more photostable than a NIR-emitting cyanine dye currently used in microscopy (DTDCI, Figure 4). Photostabilities did not depend on the
Figure 4. Comparison of the photostabilities of Nd and Yb complexes of Chl15Pd with a NIR-emitting cyanine dye, NTDCI (3,3′diethylthiadicarbocyanine iodide, λex = 649 nm). λex = 390 nm for [Yb(Chl15Pd)3]3− and [Nd(Chl15Pd)3]3−. Water/THF were used as the solvent, and the absorptions of the solutions were adjusted to the same value. The connecting straight lines are there to guide the eye.
nature of the Ln ion. This is interesting, as a Nd-free base chlorin dyad could produce singlet oxygen through chlorin triplet excited state quenching by atmospheric oxygen; the triplet state was populated by thermal energy back transfer from Nd.17 It is possible that singlet oxygen is not produced in our dyads, as the Pd chlorin triplet is higher-lying than that of the free base chlorin, which makes back energy transfer unfavored.18 The Pd chlorin antenna without the Ln decomposed rapidly under the same conditions. Therefore, it seems that the Ln rapidly and irreversibly quenches the Pd chlorin excited state and prevents its reaction with atmospheric oxygen. Finally, we turned our attention to the sensitization mechanism to understand why the red-absorbing chlorins were competent antennae. Sensitization often proceeds via the antenna triplet state.12 We studied the possibility of a singlet-mediated sensitization by comparing the residual chlorin fluorescence in Nd(Chl3FB)3, Yb(Chl3FB)3, and Gd(Chl3FB)3 (Figure S56 and S57). Gd(III) has only high-lying excited states, and thus cannot accept energy from the antenna. Compared to the Gd complex, the Nd and the Yb species had diminished chlorin fluorescence (Figure S57), which may be due to direct energy transfer from the chlorin singlet excited state. A photoredox mechanism involving electron transfer (eT) from the excited antenna to Yb(III), followed by back electron transfer to the antenna radical cation is known for Yb(III) complexes, and would also diminish the antenna fluorescence.34,35 However, eT can explicitly be ruled out in the case of chlorin antennae by
Figure 3. Multiplex detection with (a) emission-based (λex = 590 nm) and b) excitation-based discrimination (λem = 980 nm), [complex] = 9 μM for single lanthanide complex concentrations (blue and red lines), [complex] = 4.5 μM for each lanthanide complex in the mixture, [lanthanide] = 9 μM total (black line). Spectra were recorded in water/THF; the asterisk marks a peak that contains contributions from an artifact. 10977
DOI: 10.1021/jacs.8b07609 J. Am. Chem. Soc. 2018, 140, 10975−10979
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Journal of the American Chemical Society estimating the driving force for eT.36 Typical 10-aryl-substituted chlorins have first oxidation potentials at ∼1.03 V vs NHE. The Yb(II/III) redox potential for the hydrated ion is −1.15 V.37 This value is an upper estimate, as the overall 3− negative charge of the complex likely stabilizes Yb(III), and shifts the reduction to more negative potentials. The driving force for PeT from the excited singlet state of the chlorin (1.97 eV) is thus endergonic by 0.21 eV, so unfavorable (without the usually small contribution of the interaction between the Yb(II) and the chlorin radical cation). Therefore, we conclude that the efficient overlap of the chlorin singlet (∼15 000− 17 000 cm−1), and triplet (∼13 000 cm−1 for an analogous 15-Ph Pd chlorin)38 excited states, which are located
